CALIFORNIA STATE UNIVERISTY, NORTHRIDGE
Initial Studies of Isobutylamine N-Hydroxylase
(vlmH), an Enzyme in the Valanimycin Biosynthetic Pathway
A thesis submitted in partial fulfillment of the requirements
For the degree of Master of Science in Biochemistry
By
Charmaine Ibarra
May 2015
The thesis of Charmaine Ibarra is approved:
Dr. Paula Fischhaber
Date
Dr. Joseph Hajdu
Date
Dr. Jessica Vey, Chair
Date
California State University, Northridge
ii
DEDICATION
This thesis is dedicated to my parents and family
for their love and encouragement.
iii
ACKNOWLEDGMENTS
The pursuit of this research project has been an exciting journey and could not be
possible without my parents, Gus Ibarra and Sharmain Palma. This research signifies my
growth as an individual and as a biochemist; I would like to express my sincere gratitude
to my research advisor Dr. Jessica Vey for her mentorship throughout the years. The
compilation of this thesis could have not been possible without the oversight provided by
my thesis committee, Dr. Paula Fischhaber and Dr. Joseph Hajdu.
To the Vey lab members, Lily, Casper, Derek, Jaleh, Zara and Kelvin, thank you
for your incredible friendship through out the years. Lauren, thank you for your
friendship and advice throughout the vlmH project. Nancy, your assistance on the
construction of the vlmH vectors is greatly valued. Thank you Brad for your help in
generating figures in Pymol. Richard, and Lynn, your assistance in determining the
activity of vlmH is appreciated. Thank you all for memorable moments. I will cherish
your friendship and take with me the advice you have provided as I continue on to great
things.
Additionally, I would like to thank the graduate students in the Chemistry
Department; they have been a strong support system through this process. The time they
took to help me in lab, revise my thesis chapters, and listen to me practice is much
appreciated. Their constructive criticism has greatly improved my character in many
ways. I would like to acknowledge the faculty as well, Dr. Fischhaber, Dr. Crowhurst,
Dr. Medh, and Dr. Hajdu. They have instilled in me the knowledge I need to continue in
my career as a biochemist.
iv
TABLE OF CONTENTS
Signature Page…………………………………………………………………………….ii
Dedication………………………………………………………………………………...iii
Acknowledgments………………………………………………………………………..iv
List of Figures …………………………………………………………………………viii
List of Tables……………………………………………………………………………..xi
List of Abbreviations…………………………………………………………………….xii
Abstract………………………………………………..………………………………...xiii
Chapter 1: Introduction……………………………………………………………………1
Research Significance …………………………..…………………………………….1
Antibiotics and Bacterial Resistance in the 21st Century ……..………………..…1
Valanimycin: A Simple Azoxy Natural Product…………………..……………...2
The Biological Context of vlmH……….………....…...................................................4
The Valanimycin Biosynthetic Pathway…………………………………..………4
Previous Studies……………………………………………………………….......5
An Overview of the Flavin-Dependent Monooxygenases (FMOs)………..…….........6
Activation of Molecular Oxygen by Flavin…..……………………………...........6
The Catalytic Cycle……………………………………………………………..…7
One-Component vs. Two-Component………………...………………………..…9
Classification of FMOs…………………………………………………………..10
Class D Flavin-Dependent Monooxygenases……………………………..…………12
General Features………………………………………...………………….........12
Reaction Types…...………………………………………………………………13
Hydroxyphenylacetate monooxygenase, a Prototypical Class D FMO…...……..14
Motivations for Studying Isobutylamine N-Hydroxylase (vlmH)……….…………..16
Research Goals…...………………………………………………………………16
Methods used to Study vlmH…...………………………………………………..17
v
Chapter 2: Expression and Purification of vlmH...……………………….……………...19
Expression Vectors…………………………………………………………………..20
Protein Expression…………………………………………………...........................22
Major Purification Steps………………..……………………………………………24
Materials and Methods……………………………………………………………….26
Materials…………………………………………………………………………26
DNA Handling and Molecular Biology………...………………………………..27
Expression of vlmH in E.coli……………………………………………….........28
Protein Purification Methods……………………………………………….........29
Results and Discussion……………………………………………..…..……………32
Construction of vlmH Vectors.……………………………………..……………32
Expression of vlmH Vectors in E.coli……………….…………………..………35
Protein Purification…………………………………………………………..…..40
His6-vlmH………………………..……………………………………....40
His6-MBP-vlmH…………………………………………………………41
Proteolysis of His6-MBP-vlmH ………………………………………....42
Comparison of the 18°C and 25°C His6-MBP-vlmH growths…………..………44
Summary of Optimized vlmH Expression and Purification Method……..……...45
Conclusion………………………………………………………………….....…46
Chapter 3: Initiation of Biochemical and Crystallization Studies of vlmH……………...47
Methods for Detecting vlmH Activity………….……………………………………48
Methods for Crystallization Experiments……………………………………………50
Materials and Methods……………………………………………….………………54
Materials……..…………………………………………………………………..54
Assay Methods…………………………………………………………………...54
Crystallization of vlmH……………………………………………….…….........56
Results and Discussion………………………………………………..……………..58
Activity Assays
Flavin Reductase Activity Assay………………………………………...58
VlmH Catalyzes Formation of Isobutylhydroxylamine………...…..........61
Crystallization……………………………………………………………............62
Prep 1 Crystals…………………………………………………………...63
vi
Prep 2 Crystals………………………………………….………………..68
Conclusion…………………………………………………….……....…………70
Conclusion and Future Directions……………………………………………………….70
Bibliography……………………………………………………………………………..73
Appendix A: Summary of vlmH Expression Trials in E.coli……………………………78
Appendix B: Summary of Fusion Vectors Expressed in E.coli …………………………82
Appendix C: Summary of His6-vlmH Purification……………………………...……….87
Appendix D: Summary of Prep 1 Crystallization Trials…………………………………88
vii
LIST OF FIGURES
Figure 1: Structures of valanimycin and its ammonia adduct……………………………..2
Figure 2: Azoxy compounds ……………………………………………….………....…..3
Figure 3: The valanimycin biosynthetic pathway of S. viridifaciens ………………..……4
Figure 4: Flavin, an essential cofactor…………………………………………………….6
Figure 5: The reaction of reduced flavin with O2…………...…………………………….7
Figure 6: Reduction of the flavin cofactor by NADH………………………………….....8
Figure 7: General mechanism of oxygenation reactions catalyzed by FMOs………….....9
Figure 8: Oxygenation reactions catalyzed by 3 members of the FMOs……...………....11
Figure 9: Crystal structures of HPAH………………………………….………………...13
Figure 10: Hydroxylation and N-hydroxylation reactions by class D FMOs……………14
Figure 11: Reaction catalyzed by prototypical Class D FMO HPAH …………………..15
Figure 12: The active site structure of HPAH from A. baumannii……………………....16
Figure 13: Genetic features of a vector…………………………………………………..20
Figure 14: BamHI and XhoI cleavage sites for subcloning..…………………………….21
Figure 15: A standard E.coli growth curve………………………………………………23
Figure 16: Illustration of the N-terminal tag interacting with Ni2+ resin………………...26
Figure 17: Digestions of pUC57-vlmH, pLM302 and pETSUMO……………………...32
Figure 18: Double digestion of pGEX-KG-vlmH……………………………………….33
Figure 19: Digestion of pETSUMO-vlmH and pLM302-vlmH…………………………34
Figure 20: SDS-PAGE analysis of pBG100-vlmH expression in pLysS………………..35
Figure 21: SDS-PAGE analysis comparing pET24(+)-vlmH expression in three different
E.coli strains….37
Figure 22: Amino acid sequence for the fusion protein His6-MBP-vlmH……………….39
viii
Figure 23: SDS-PAGE analysis of pBG100-vlmH expression in pLysS and affinity
purification…...40
Figure 24: UV Trace of Ni2+ column purification of His6-MBP-vlmH …………………42
Figure 25: UV Trace of Ni2+ column purification of vlmH…….……………………..…43
Figure 26: SDS-PAGE analysis of His6-MBP-vlmH purification….……………………44
Figure 27: Co-purification of flavin with His6-MBP-vlmH……………………………..45
Figure 28: Absorbance spectra of His6-MBP-vlmH and oxidized flavin………….…….45
Figure 29: Detection method used to determine the concentration of IBHA……………49
Figure 30: Derivatization of an amine with NBD-Cl…………………………………….50
Figure 31: The hanging drop vapor diffusion method……………………..…………….51
Figure 32: Phase diagram of protein crystallization……………………………………..52
Figure 33: FMN quickly reduces to FMNH2 and is maintained in its reduced form……59
Figure 34: Flavin reductase assay with the addition of vlmH and no substrate present…60
Figure 35: Flavin reductase assay with the addition of vlmH and substrate present…….60
Figure 36: Reaction rate increases with vlmH concentration……………………………62
Figure 37: VlmH needles obtained from Crystal Screen HR2-110……………………...63
Figure 38: Sample crystal optimization grid screen……………………………………..64
Figure 39: Compiled images from the Additive Screen HR244…………………………65
Figure 40: Comparison of needles before and after additive screen……………………..66
Figure 41: Three different seed dilutions were used to initiate nucleation………………66
Figure 42: Results of three protein crystal tests………………………………………….68
Figure 43: VlmH three-dimensional crystals obtained from Crystal Screen HR2-110.…69
Figure 44: Sample crystal optimization grid screen……………………………………..69
Figure 45: Prep 2 crystal birefringence…………………………………………………..70
Figure 46: SDS-PAGE analysis of pBG100-vlmH, vector 2, in pLysS…………………78
ix
Figure 47: SDS-PAGE analysis of pBG100-vlmH in CodonPlus……………………….79
Figure 48: SDS-PAGE of pBG100-vlmH expressed in BL21 (DE3)……………………79
Figure 49: SDS-PAGE of pBG100-vlmH expressed in BL21 (DE3) at 18 °C……….…80
Figure 50: SDS-PAGE analysis comparing pET21b-vlmH expression in three different
E.coli strains………..81
Figure 51: SDS-PAGE analysis of GST-vlmH expression in BL21 (DE3)……………..82
Figure 52: SDS-PAGE analysis of MBP-vlmH expression in BL21 (DE3)…………….83
Figure 53: SDS-PAGE analysis of SUMO-vlmH expression in BL21 (DE3)…………..83
Figure 54: SDS-PAGE analysis of MBP-vlmH expression in BL21 (DE3) 37 °C……...84
Figure 55: SDS-PAGE analysis of MBP-vlmH expression in BL21 (DE3) 25 °C….…..85
Figure 56: SDS-PAGE analysis of MBP-vlmH expression in BL21 (DE3) 18 °C…..….86
Figure 57: SDS-PAGE analysis of pBG100-vlmH expression in pLysS and affinity
purification……………………………………………………………………………….87
Figure 58: Optimization of Prep 1. The first two optimizations resulted in fine granular
precipitate………….88
Figure 59: Compiled images from Row A (Microseed)……………...………………….89
Figure 60: Compiled images from Row B (Microseed)………………...……………….90
Figure 61: Compiled images from Row C (Microseed).…………………...……………91
Figure 62: Compiled images from Row D (Microseed).…………………...……………92
Figure 63: Compiled images from Crystallization Under Oil (0.05 M)…………………93
Figure 64: Compiled images from Crystallization Under Oil (0.075 M)………………..94
x
LIST OF TABLES
Table 1: Relative activity of amine substrates with vlmH…………………...……………6
Table 2: Overview of the FMOs………………………………………………………....12
Table 3: Summary of all expression vectors constructed for the expression of vlmH..…22
Table 4: Lysis buffers used to resuspend harvested cell pellets…………………………29
Table 5: Buffers used to purify His6-MBP-vlmH………………………………………..31
Table 6: A summary of the initial vlmH constructs expressed in E.coli………………...38
Table 7: Raw data and calculated results from selected vlmH assays…………………...61
xi
LIST OF ABBREVEIATIONS
ACAD
Acyl-CoA Dehydrogenase
Amp
Ampicillin
Au
Absorbance Unit
BMER
β-Mercaptoethanol
CV
Column Volume
DTT
Dithiothreitol
E.coli
Escherichia coli is a Gram-negative bacterium
FMO
Flavin-dependent monooxygenase
FPLC
Fast Protein Liquid Chromatography
GST
Glutathione S-transferase
HPAH
4-hydroxyphenylacetate 3-hydroxylase, a Class D FMO
IPTG
Isopropyl β-D-1-thiogalactopyranoside
KAN
Kanamycin
LB
Lysogeny Broth
MBP
Maltose Binding Protein
OD
Optical Density
pETSUMO-vlmH
Gene fragment for the fusion protein His6-SUMO-vlmH
pGEX-KG-vlmH
Gene fragment for the fusion protein His6-GST-vlmH
pLM302-vlmH
Gene fragment for the fusion protein His6-MBP-vlmH
SDS-PAGE
Sodium dodecyl sulfate - Polyacrylamide gel electrophoresis
SUMO
Small ubiquitin like-modifier
pBG100-vlmH
Gene fragment for the protein His6-vlmH
UV
Ultraviolet lamp
xii
ABSTRACT
Initial Studies of Isobutylamine N-Hydroxylase
(vlmH), an Enzyme in the Valanimycin Biosynthetic Pathway
By
Charmaine Ibarra
Master of Science in Biochemistry
Isobutylamine N-hydroxylase (vlmH) is a class D flavin-dependent
monooxygenase (FMO) in the valanimycin biosynthetic pathway of Streptomyces
viridifaciens. Valanimycin is a relatively simple azoxy antibiotic that exhibits antitumor
and antibacterial properties. VlmH uses reduced flavin to insert an oxygen atom into
isobutylamine (IBA), which yields the hydroxylated product, isobutylhydroxylamine
(IBHA). IBHA is coupled to serine and, after a series of steps, is converted to the final
product. Interest in the biosynthetic pathways of antibiotics like valanimycin is increasing
lately due to the increasing prevalence of antibiotic resistant bacteria. In our efforts to
develop new therapeutics, researchers are beginning to focus on understanding and
engineering pathways that synthesize natural products with therapeutic activity. Enzymes
like vlmH are found in numerous natural product pathways; by investigating their
reactivity, we can enable bioengineering of those biosynthetic pathways.
VlmH was expressed, purified, and assayed in previous studies; however, no
structural information is available yet and the detailed catalytic mechanism is unknown.
In this research project, we aim to establish reliable methods for purifying and assaying
vlmH. We will use these methods to study enzymatic catalysis and determine the
xiii
enzyme’s X-ray crystal structure. We began by constructing vlmH expression vectors to
determine which vector yields soluble vlmH. Several alternatively tagged constructs of
vlmH were generated. Use of a maltose binding protein (MBP) fusion system results in
soluble expression of vlmH at two induction temperatures. Our current purification
strategy yields on average 4.08 mg vlmH per liter culture (with induction at 25 °C) and
2.10 mg vlmH per liter culture (with induction at 18 °C).
To assay the activity of vlmH, we replicated a spectrophotometric assay
developed for this enzyme by Ronald Parry. Reduction of Fe(III) by the hydroxylamine
product is detected by formation of a complex between Fe(II) and 2,4,6,tripyridyl-striazine. The concentration of this species can be determined spectrophotometrically and
is proportional to the concentration of IBHA. Our experiments show that the amount of
product formed in an assay increases with vlmH concentration, suggesting that our vlmH
is active. This indicates that the protein is properly folded, a prerequisite for
crystallization studies. Broad crystallization screens identified conditions that led to two
crystal forms of vlmH. Optimization of the conditions are ongoing in order to obtain high
quality crystals, which are necessary for X-ray diffraction.
With these methods in hand, our lab can now pursue more detailed experiments
focused on understanding the structure and function of vlmH.
xiv
CHAPTER 1
INTRODUCTION
Research Significance
Antibiotics and Bacterial Resistance in the 21st Century
Bacterial resistance is recognized as a public health concern.1,2,3 As bacteria
continue to develop antibiotic resistance, the number of therapeutics we can turn to for
effectively treating resistant infections decreases. 4,5,6 Between 1940 and 1962, more than
20 new classes of antibiotics were discovered. 7,8 To date, only two new classes of
antibiotics have been marketed.
Anyone can be affected by a drug-resistant infection, the young, old and
chronically ill are all at risk. It has been reported that 99,000 deaths result from
antibacterial-resistant pathogens.3 Common therapeutics like, vancomycin, penicillin and
quinolones are losing effectiveness against multiple types of bacteria.9 As resistance
increases, and as bacteria trade resistance genes, this is going to become a bigger and
bigger problem. The cost of treating resistant infections will increase into the billions.3,9
With an emerging threat of serious infection by antibiotic resistant bacteria, there
has been a renewed interest in bacterial secondary metabolic pathways that synthesize
therapeutic compounds. One current area of interest in the antibiotic development arena
is metabolic engineering of bioactive naturally available therapeutic compounds.
Studying the chemical reactivity of the individual enzymes of those antibiotic
biosynthetic pathways (such as vlmH of the valanimycin pathway) should enable largerscale metabolic engineering projects.
1
Valanimycin: A Simple Azoxy Natural Product
Valanimycin is an azoxy antibiotic (Figure 1). Masayuki Yamato and Hamao
Umezawa first isolated it as a colorless, soluble, unstable oil.10 It was isolated and
structurally characterized as the stable ammonia derivative shown in Figure 1.
Figure 1: Structures of valanimycin and its ammonia adduct. The compound contains
a relatively rare azoxy group, highlighted in red in this figure. (a) The nitrogens of the
azoxy group are thought to be derived from the amines of valine and serine.
(b)Valanimycin exists as an unstable oil if dried in the absence of salt. Ammonium
hydroxide (NH4OH) was used to obtain the ammonia adduct.
Yamato and Umezawa also found that the valanimycin ammonia adduct has antibacterial
and antitumor properties. It can inhibit the growth of gram-positive and gram-negative
bacteria as well as mouse leukemia cells, and the life span of mice with Ehrlich
carcinoma was extended by administration of the molecule.10
In E. coli BE1121, a bacterial strain incapable of repairing its DNA, the presence
of valanimycin inhibits cell growth, suggesting that valanimycin targets some aspect of
DNA synthesis or repair.10 Incorporation studies were used to determine the exact
cellular process targeted by valanimycin. Four different radioactive biological precursors
were incubated with E. coli BE1121, in the presence of valanimycin. Radiolabeled
2
acetyl-D-glucosamine (a structural component in bacterial cell walls), labeled leucine,
and labeled uridine were all incorporated properly into their respective cellular
macromolecules, suggesting that cell wall synthesis, protein synthesis and transcription
are not targeted by the molecule. However, [6-3H]thymidine was not incorporated into
DNA in the presence of valanimycin, supporting the proposal that valanimycin interferes
with DNA synthesis.5
The azoxy group is present in a growing class of natural products (Figure 2).
Though this unique functional group is exhibited by a number of biologically active
compounds, the formation process of the moiety remains unknown. By studying enzyme
structure and function, we can compare similarities between enzymes that catalyze key
steps in the synthesis of the azoxy group.
Figure 2: Azoxy compounds. Natural compounds containing the characteristic azoxy NN bond.11,12,13,14 The stability, toxicity, and clinical applications has not been reported.
3
The Biological Context of vlmH
The Valanimycin Biosynthetic Pathway
Isobutylamine N-hydroxylase (vlmH) is a class D flavin-dependent
monooxygenase (FMO). FMOs incorporate a single oxygen atom into an organic
compound using flavin to activate and reduce the oxygen (vide infra). VlmH catalyzes
the N-hydroxylation of isobutylamine (IBA) to isobutylhydroxylamine (IBHA) in the
Valanimycin biosynthetic pathway shown in Figure 3.
Figure 3: The valanimycin biosynthetic pathway of S. viridifaciens. Valanimycin is
synthesized from valine and serine starting materials.15,16 The hydroxylation reaction
catalyzed by vlmH is boxed in green. Pathway from reference 17.
4
The monooxygenase vlmH and the reductase vlmR catalyze the insertion of one
atom of oxygen into IBA. The hydroxylated product IBHA is attached to serine and
through a series of enzymatic steps is converted to valanimycin.
Previous Studies
Throughout the 1990s, Ronald J. Parry’s research (carried out at Rice University)
focused on understanding formation of the azoxy group found in the natural products
shown above (Figure 3). He chose to study valanimycin in detail due to its smaller size.
From the 1990s to the present, Parry elucidated part of the biological pathway, which
includes 14 enzymes involved in the biosynthesis and regulation of the pathway.17 As
part of their studies, they published methods for expression, purification, and enzymatic
assay of vlmH and several other enzymes in the valanimycin pathway.17-22
Parry and Li determined that two enzymes are required for hydroxylation of IBA,
which is further modified in downstream steps to yield the final valanimycin product. The
first enzyme, vlmR18, provides reduced flavin for the second enzyme, vlmH, which
catalyzes the hydroxylation reaction.19,20,21 Parry showed that reduced flavin and
molecular oxygen were required for catalysis. According to vlmH’s catalytic
requirements and sequence homology, vlmH is a member of the flavin-dependent
monooxygenase superfamily, a group of enzymes that use flavin to catalyze the
incorporation of oxygen.22 Experiments on vlmH substrate selectivity are summarized in
Table 1 and show that vlmH exhibits some substrate promiscuity.20 Additionally, it
appears that vlmR may transfer flavin directly to vlmH rather than allowing free diffusion
of the cosubstrate.18,20 This may influence substrate selectivity of vlmH in a manner that
is not yet understood.20
5
Table 1: Activity of Amine Substrates with vlmH and the NAD(P)H:FMN
Oxidoreductase of V.fischeri.
Amine
Relative Activitya
Isobutylamine (IBA)
1.0
n-Propylamine (NPA)
1.25
n-Butylamine (NBA)
0.87
Sec-Butylamine (SBA)
0.39
Benzylamine (BZA)
0.61
a
The value was an average of three determinations using the spectrophotometric assay.
Table from reference 20.
An Overview of the Flavin-Dependent Monooxygenases (FMOs)
Activation of Molecular Oxygen by Flavin
FMOs exist in a variety of biological pathways and catalyze the insertion of one
atom of oxygen from molecular oxygen into an organic substrate. FMOs require a flavin
cofactor, either flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD),
shown in Figure 4.
Figure 4: Flavin, an essential cofactor. Isoalloxazine is the tricyclic organic ring of the
flavin nucleotides, where two hydrogen atoms are accepted or donated. FMN and FAD
differ in the presence or absence of adenosine monophosphate, as indicated. From
reference 23.
6
The flavin cofactor is a ubiquitous molecule that is capable of a range of redox
chemistry, including activation and reduction of molecular oxygen. Oxygen insertion
reactions using O2 and organic molecules are generally spin forbidden.24 Molecular
oxygen is a triplet in its ground state, making reactions with singlet molecules (such as
those catalyzed by the FMOs) difficult due to spin conservation in the reactants and
products – most organic radicals are not particularly stable, making these reactions
unfavorable.25, 26 Unlike most organic molecules, reduced flavin can react with the
molecular oxygen triplet to yield a stable radical – the one-electron-reduced form of the
cofactor, the flavin semiquinone radical. 27 Therefore, flavin can be used to overcome this
spin barrier and promote reaction of an organic molecule with oxygen because the flavin
semiquinone produced is sufficiently stable. 28,29 Figure 5 summarizes this reaction.
Figure 5: The reaction of reduced flavin with O2. The first step in the reaction of a
reduced flavin with O2 is a single electron transfer to form the caged radical
semiquinone-superoxide pair. From reference 24 and 25.
The Catalytic Cycle
The overall catalytic cycle of FMOs consists of two half-reactions, the reductive
and oxidative half reactions.24-27 In the reductive half-reaction oxidized flavin is reduced
by nicotinamide adenine dinucleotide (NADH) as shown in Figure 6. The reduction step
can be carried out by a dedicated flavin reductase, or by the monooxygenase itself,
depending on the enzyme system.28-31 Transfer of flavin from a reductase to the site of
7
monooxygenation, if necessary (see below), can occur by diffusion or by direct transfer
between enzymes.24, 27-31
Figure 6: Reduction of the flavin cofactor by NADH. Hydride is transferred from
NADH to the N5 position on the isoalloxazine ring.
The oxidative half-reaction begins with the reaction between the hydroquinone form of
flavin and O2 to form the semiquinone form and superoxide radical (see Figure 5). The
caged radical pair combine to form the peroxyflavin intermediate (Figure 7), which is
followed by oxygenation of the substrate. The electron withdrawing isoalloxazine ring
plays a critical role in the mechanism of oxygen transfer to the organic substrate. The
electronegative ring withdraws electrons through the sigma bonds and polarizes the O-O
bond, thereby activating it for cleavage.24-27 Depending on the protonation state of flavin,
an organic substrate can react by nucleophilic or electrophilic oxygenation (Figure 7).
8
Figure 7: General mechanism of oxygenation reactions catalyzed by FMOs. The
electron rich isoalloxazine ring activates O2. A covalent bond is formed between O2 and
C4a of the ring, yielding peroxyflavin or hydroperoxyflavin. The hydroperoxyflavin is
relatively unstable but FMOs are able to stabilize the species and use it to oxygenate a
substrate. ‘X’ represents an organic substrate. From references 24, 28, 31, and 33.
One-Component vs. Two-Component System
FMOs can be classified as one-component or two-component systems based on
how they obtain reduced flavin for catalysis. 30,31 One-component FMOs reduce flavin
and catalyze the oxygenation of their organic substrate in the same active site. In contrast,
two-component FMOs consist of two distinct enzymes: a reductase (often called an
“external reductase”) to reduce flavin and a monooxygenase (also called a
“hydroxylase”) that catalyzes the oxygenation reaction.26 The electron donor for both
systems is NAD(P)H. In addition, the cofactor preference (FAD vs. FMN) of each system
varies depending on the FMO class and the individual enzyme.21,26
9
Two-component systems use external reductases to catalyze a redox reaction
between flavin and the electron donor. After NADH and flavin bind to the flavin
reductase, two electrons and one proton are transferred to the isoalloxazine ring at atom
N5, followed by protonation at N1 (see Figure 6). The final product of this redox reaction
is reduced flavin.17-32
Classification of FMOs
FMOs all use reduced flavin to activate molecular oxygen and catalyze
oxygenation reactions.18,22 The type of oxygenation reaction and substrate depends on the
shape and chemical environment of the active site of each FMO.26 The individual
enzymes, though, vary in several ways. The FMO family is divided into classes A
through H based on cofactor preference, structural and sequence homology and type of
oxygenation reaction.23,24,26 The eight classes perform oxygenation reactions on a wide
variety of substrates. Representative oxygenation reactions catalyzed by three of the eight
classes are shown in Figure 8 and general features of the FMO classes are summarized in
Table 2.
10
Figure 8: Oxygenation reactions catalyzed by three of the eight classes in the FMO
enzyme family.
There are many ways to classify FMOs, Table 2 summarizes the classes based on
cofactor preference, protein fold and the type of oxygenation reaction catalyzed.
11
Table 2: Overview of FMOs.
Class
Cofactor
Electron Donor
Protein Fold
Reaction
A
FAD
NAD(P)H
Rossmann
B
FAD
NAD(P)H
Rossmann
C
FMN
FMNH2
D
E
F
FAD/FMN
FAD
FAD
NADPH/NADH
FADH2
FADH2
Tim-barrel
Acyl-CoA
dehydrogenase
Rossmann
Rossmann
G
FAD
Substrate
Rossmann
H
FMN
Substrate
Tim-barrel
Hydroxylation
Sulfoxidation,
N-hydroxylations and
others
Baeyer-Villiger
Oxidation, Epoxidation,
Desulfurization,
Sulfoxidation
Hydroxylation,
N-hydroxylation
Epoxidation
Halogenation
Oxidative
Decarboxylation
Oxidative
Decarboxylation
The cofactor and electron donor preference varies among the classes. Class C is the most
diverse, catalyzing five different types of oxygenations. Reaction types are attributed to
protein folds.33 Table adapted from reference 33.
Class D Flavin-Dependent Monooxygenases
General Features
Class D FMO systems have several common features. Class D FMOs are twocomponent systems, consisting of a monooxygenase that catalyzes the insertion of
molecular oxygen into substrate, which requires reduced flavin produced by an external
flavin reductase.30-31 The currently known class D FMOs catalyze electrophilic aromatic
substitutions and N-hydroxylation reactions with their cofactor preference (FAD vs.
FMN) varying from enzyme to enzyme.31,33 The monooxygenase components of this
class of enzymes have some sequence homology (20-30 % identity) to the acyl-coA
dehydrogenases, a large family of flavin-dependent enzymes involved in fatty acid
catabolism.31,33 The first class D FMO family structures showed that the enzymes adopt
12
the acyl –CoA dehydrogenase (ACAD) tertiary fold. Enzymes that adopt the ACAD fold,
which is illustrated in Figure 9, are homotetrameric proteins formed by oligomerization
of monomers with three-domains of primarily α-helical secondary structure.28,31,33 The
ACAD fold can be described as catalytically diverse because the fold appears in enzymes
that catalyze hydroxylations, dehydrations, and oxidations.
Figure 9: The crystal structure of HPAH. The prototypical Class D flavin
monooxygenase, 4-hydroxyphenylacetate 3-hydroxylase (HPAH) is a homotetramer (a)
and adopts the ACAD fold (b). Each monomer is shown in a distinct color. This figure
was generated in Pymol. PDB ID: 2YYG. From reference 34.
Reaction types
Like the class A flavin monooxygenases, class D FMOs have relatively narrow
substrate selectivity and demonstrate relatively high regio- and enantioselectivity. At
13
present, the reactions catalyzed by the known class D FMOs fall into two categories:
electrophilic aromatic substitution (illustrated by HPAH)34 chlorophenol-4monooxygenase (TftD)35 and para-nitrophenol 4-monoxygenase (PnpA)36,37 and Noxygenation (illustrated by vlmH)19,20,22 ORF3638, KijD339 and DnmZ40. An example of
each reaction type is shown in Figure 10. In the representative enzymes that catalyze
electrophilic aromatic substitutions, the oxygen insertion typically takes place at the
ortho- and para- positions because these sites are electron donating on a ring substituent.
Figure 10: The class D FMOs catalyzes hydroxylation and N-hydroxylation reactions on
aromatic and non-aromatic substrates.
Hydroxyphenylacetate monooxygenase, a Prototypical Class D FMO
Several class D FMOs have been biochemically and/or structurally
characterized.41,42 The first class D member to be identified and studied in detail was 4hydroxyphenylacetate 3-hydroxylase (HPAH). 31,33,34 HPAH catalyzes the hydroxylation
14
of p-hydroxyphenylacetate (HPA) to 3,4-dihydroxyphenylacetate (DHPA), shown in
Figure 11.
Figure 11: The reaction catalyzed by prototypical Class D FMO monooxygenase 4hydroxyphenylacetate 3-hydroxylase (HPAH) from Acinetobacter baumannii.
HPAH represents the model enzyme for studying the catalytic mechanism of class D
FMOs, including the method of flavin transfer and regulation. Two class D HPAHs have
been characterized, one in Acinetobacter baumannii and the other in Pseudomonas
aeruginosa.31,33 The crystal structure of HPAH from A. baumannii has provided a
structural framework to understand the catalytic mechanisms of class D FMOs in general,
and the kinetics of HPAH in particular. 31,33 Studies following the formation of the flavin
C4a-hydroperoxide intermediate from the A. baumannii system have confirmed the roles
of specific residues involved in catalysis.
Two residues, His 396 and Ser171, are important for the formation and
stabilization of the C4a-hydroxyperoxy flavin.41,42 His-396 is involved in the formation of
the C4a-hydroperoxyflavin intermediate as shown by stopped flow experiments. In
addition, His-396 activates the substrate for electrophilic substitution through interactions
with the phenolate form of the substrate. The hydroxyl side chain of Ser-171 hydrogen
bonds with the N5 atom of the isoalloxazine ring. This interaction appears to stabilize the
flavin C4a-hydroperoide species to hydroperoxide elimination. Figure 12 shows the
15
active site of the monooxygenase, highlighting the position of S171 and H396 with
respect to the flavin isoalloxazine ring.
Figure 12: The active site structure of HPAH from A. baumannii. The hydroxyl group
of Ser171 interacts at the N5 atom of the isoalloxazine ring. H396 interacts with the
(-OOH) that forms at the C4a atom. Figured generated in Pymol. PDB ID: 2JBS. From
reference 42.
His120 has also been suggested to behave as a general acid during the electrophilic
aromatic substitution.42 These three functions, and those of His-396 and Ser-171 in
particular, are likely conserved in all the class D flavin monooxygenases, though the
details may vary depending on the individual reaction catalyzed.
Motivations for Studying Isobutylamine N-hydroxylase (vlmH)
Research Goals
Our first research project goal was to establish methods to obtain and
experimentally characterize vlmH. These methods will be used to carry out a more
detailed kinetic analysis of the enzyme and structural characterization by X-ray
16
crystallography. Though the expression, purification, and activity of vlmH have been
reported, the enzyme has not been crystallized.20 Therefore, the goals of the vlmH project
are to 1) construct expression vectors to express vlmH with an N-terminal tag, 2) express
and purify vlmH, 3) show that vlmH is active towards IBA, and 4) crystallize vlmH.
Three of these goals (1-3) have been completed, and the methods have been used to
purify two vlmH mutants (S158A and H356A) as well. Our future steps include studies
on the pH and temperature dependence of enzyme activity. A crystal structure will allow
us to verify predictions on active site residues and study the flavin monooxygenase fold
in more depth.
Overall, we are interested in studying vlmH as a flavin-dependent protein; this
class of enzymes demonstrates wide chemical reactivity.
Methods Used to Study VlmH
A substantial amount of research has been dedicated to understanding the
biosynthesis of the azoxy group. The methods used to study vlmH previously include
protein expression in E.coli, purification and biochemical assays. While those previous
experiments have given insight to the activity of vlmH, X-ray crystallography can
provide structural information at atomic resolution. Information such as active site
residues involved in catalysis and conformational changes that occur upon ligand binding
can be identified and/or confirmed with a molecular-level model of the protein.
Therefore, a series of structures of vlmH is a major future goal of our research lab. To
reach this goal we need a reliable source of active, pure vlmH. Our first step was to
develop methods to produce and assay vlmH – each method is described in detail in the
following chapters.
17
Bacterial expression systems are good options for producing the high yields of
vlmH required for activity assays and crystallization experiments. An E. coli expression
system provides many advantages for expression of bacterial proteins: high protein yields
can be achieved and, because this organism is very well-understood, there are wellestablished methods available for all aspects of the process, from bacterial culture to
protein production to troubleshooting. Our vlmH expression constructs are unique;
therefore, protocols for expression and purification needed optimization.
After an E. coli expression system produces large amounts of a recombinant
protein, that protein must be isolated from the thousands of native E. coli proteins.
Affinity chromatography, a technique that exploits specific binding interactions between
molecules, was used to separate vlmH from these native proteins. Our lab uses a fast
protein liquid chromatography (FPLC) system to isolate vlmH, ultimately yielding a
high-purity sample.
The typical experimental step following purification of a recombinant enzyme is
confirmation that the enzyme is active. The assay used here to quantitate vlmH activity is
adapted from Parry and Li. Their indirect assay method allows for the quantitation of the
amount of product formed over a given time period.20 With confirmed enzyme activity,
crystallization experiments follow. These biochemical studies will provide a more
detailed understanding of the structure and function of vlmH.
18
CHAPTER 2
EXPRESSION AND PURIFICATION OF
ISOBUTYLAMINE N-HYDROXYLASE (VLMH)
Introduction
Biochemical assays and crystallization experiments require a pure and highly
concentrated (>5 mg/mL) protein sample. Our vlmH production strategy uses expression
in E. coli using a maltose binding protein tagged form of vlmH and purification by
affinity chromatography to produce the pure and concentrated samples required. Utilizing
E. coli as an expression system allows us to produce large quantities of vlmH which can
then be purified away from other cellular proteins. We chose to use affinity tags (a 6residue histidine tag, in particular) and affinity chromatography to achieve purification of
vlmH.
Three variables affect the production of soluble protein: the expression vector,
type of E. coli strain, and induction conditions. The first part of this research focuses on
each variable with the objective to identify the best conditions to establish a protocol for
expression and purification of vlmH. Among six vectors created for this study, the vector
pLM302-vlmH, a maltose binding protein (MBP) fusion system, resulted in soluble
expression of vlmH. When paired with a reduced induction temperature, this resulted in a
high yield of vlmH in the cell line E. coli BL21 (DE3).
This chapter describes the methods used to prepare vlmH for expression in E.
coli. The steps were: 1) construction of expression vectors with the vlmH gene 2) growth
of bacterial cultures to assess the yield and solubility of each vlmH vector and 3) the
large-scale production and purification of vlmH.
19
Expression Vectors
Expression vectors, also known as constructs or plasmids, are used for protein
expression in cells. Vectors consist of DNA, a gene insert (vlmH gene), and several
essential genetic features. Three specific genetic features are present on typical
expression vectors. The origin of replication allows a plasmid to be replicated and
propagated in E.coli cells. The selection gene confers resistance to a specific antibiotic on
the E. coli cells that have taken up the plasmid. This allows cells transformed with the
plasmid to propagate in media supplemented with the corresponding antibiotic, enabling
selection. The last feature, the multiple cloning site (MCS), is a segment of DNA which
contains restriction sites used for in-frame insertion of the gene of interest into the vector
(Figure 13). Some vectors encode optional N- or C-terminal tags for purification,
solublization, or other experimental requirements.
vlmH
5ʹ
3ʹ
N-Terminal tag MCS
Figure 13: Genetic features of a vector. An E. coli cell containing native DNA and a
circular expression vector. The MCS is used to insert the vlmH gene next to the Nterminal tag.
DNA can be manipulated through the use of restriction enzymes, phosphatases,
ligases, and other enzymes. Using such techniques, recombinant genes like our vlmH
DNA can be introduced into a new, specific vector. A technique called subcloning is used
20
to move gene inserts from one vector to another in order to construct a specific vector. To
subclone the vlmH gene, the restriction enzymes XhoI and BamHI cut a specific
sequence of DNA within the MCS of the parent and target plasmids (pUC57-vlmH).
Digestion forms “sticky ends” shown in Figure 14, which can then be ligated with a new
DNA sequence with complementary sticky ends. In this study, the vlmH gene was
subcloned into six different vectors with BamHI and XhoI cleavage sites.
Figure 14: BamHI and XhoI Cleavage Sites for Subcloning. Restriction enzymes
recognize the colored DNA sequence and “cut” the vector, pLM302. The digestion
results in complimentary DNA end pieces. The vlmH gene contains the corresponding
DNA end pieces; the vlmH gene can be inserted into the vector pLM302, the final gene
segment is pLM302-vlmH.
The six expression vectors used in this study vary in terms of the affinity tag or
fusion partner (protein) present (see Table 3 for a summary of these vectors). Protein tags
can be used to enhance solubility and promote the proper folding of their partners
(vlmH). Examples of commonly used protein tags include maltose-binding protein (MBP,
MW = 40 kDa), glutathione S-transferase (GST, 26 kDa) and small-ubiquitin like
modifier protein (SUMO, 50 kDa). MBP facilitates proper folding and increases
solubility of the fusion partner.43 GST is designed for high-level intracellular expression
of a fusion partner.44 SUMO proteins can enhance soluble expression of proteins and
possibly decrease proteolytic degradation, which is not achieved in traditional expression
21
systems.45 All three protein tags are covalently attached to vlmH and can be removed by
a specific protease.
Table 3: Summary of all the expression vectors generated and tested for high and
soluble vlmH expression.
Expression
Vector
N-terminal
Tag
Expected
Molecular
Weight
(kDa)
Promoter
Selection
Gene
pBG100-vlmH
His6
42.88
Lac
Kanamycin
pET-21b-vlmH
pET-24(+)-vlmH
T7
none
39.83
39.83
T7
T7
Kanamycin
Kanamycin
pLM302-vlmH
His6-MBP
84.83
Lac
Kanamycin
pGEX-KG-vlmH
His6-GST
66.12
Lac
Ampicillin
pET-SUMO-vlmH
His6-SUMO
50.83
Lac
Kanamycin
Cleavage
Site
PreScission
Protease
None
PreScission
Protease
Thrombin
ULP-1
Protease
Protein Expression
E. coli Cell Lines
Our vlmH vectors are designed for recombinant protein expression in E. coli. In
this study, three E. coli strains [BL21(DE3), BL21(DE3)-pLysS, and BL21(DE3)CodonPlus] were compared to determine which gave the highest protein yield. BL21
(DE3), designed for high-level induction and expression of non-toxic bacterial genes, is
the reasonable strain to use for vlmH based on the cell line’s features. The other two
strains include features we do not expect to need for vlmH expression, (pLysS is
optimized for expression of toxic genes, while CodonPlus encodes tRNAs for rare
codons, which were removed from our vlmH gene through codon-optimization). Still, all
three strains were tested in the off chance that one might unexpectedly outperform the
others.
22
Induction of Protein Expression
Our vlmH vectors contain either a lac operon or T7 promoter, and protein
expression is induced by addition of the lactose derivative, isopropyl-β-D-thiogalactoside (IPTG). When IPTG is present in the cell, it binds the lac repressor protein,
inducing a conformational change that abrogates repressor DNA binding and allows
transcription. The T7 promoter functions just like the lac operon. When IPTG is present
in the cell, E. coli synthesizes the protein T7 RNAP. The T7 RNAP protein binds to the
T7 RNAP promoter and initiates transcription of the vlmH gene.46 IPTG is added to cell
cultures during the log phase shown in the E. coli growth curve in Figure 15. In the log
phase of growth, cells are dividing rapidly, increasing the potential to obtain the
maximum amount of protein.
Figure 15: A standard E. coli growth curve. The three phases are determined by
measuring the absorbance of a culture sample at A600nm. Absorbance is graphed over
time. Cell density increases slowly in the lag phase; the log phase is where exponential
growth occurs. In the log phase, cells are healthy and at an ideal stage to synthesize
proteins. The stationary phase is where the medium’s nutrients become limiting and
waste accumulates to deleterious levels. From reference 47.
23
Induction Temperatures
E. coli’s optimal growth temperature is 37 °C. Bacterial doubling time and protein
expression are most efficient at this temperature, and so it is the standard temperature
used for E. coli culture and induction of protein expression. However, recombinant
protein expression at too fast a rate can lead to protein misfolding and aggregation to
form inclusion bodies. In such cases, lower temperatures are often used during the
induction period to slow the rate of protein expression and thereby decrease inclusion
body formation.48
Protein Expression Trials
To determine the best induction time and temperature for vlmH expression,
expression trials were performed for each vlmH vector. For each expression trial, samples
of cell culture are collected before induction with IPTG (the pre-induction sample) and
during the induction period (post-induction sample). The pre- and post-induction samples
allow us to verify that vlmH is synthesized after IPTG is added (and not before) and see a
clear picture of the native protein content of the cells. Cell lysis supernatant and pellet
samples (see below) are also collected during an expression trial, to determine whether
the vlmH produced is soluble (appearing in the supernatant) or misfolded and expressed
in inclusion bodies (appearing in the pellet).
Major Purification Steps
The major steps involved in purification of any recombinant protein are cell lysis
and separations to remove cellular proteins. When necessary, affinity tags or fusion
proteins are removed by proteolysis and an additional separation step. Our vlmH
24
purification strategy uses lysozyme and sonication for cell lysis, affinity chromatography
as the sole separation method and proteolysis by the enzymes listed in Table 3.
Cell lysis can be accomplished in a number of different ways, including lysozyme
digestion, sonication, freeze/thaw cycles and other physical or chemical methods.49,50
Digestion by lysozyme is a physical / chemical method in which added lysozyme
degrades the bacterial cell wall.51,52 Upon weakening of the cell wall, the cells will burst
due to osmotic pressure.49-52 Sonication is a physical method for lysing cells through
agitation of the resuspended cells. High frequency sound waves (>20 kHz) cause
cavitation within the liquid suspension, which generates enough shear force to lyse cells
by disrupting their membranes. Cell lysis results in the release of vlmH, E. coli proteins,
DNA, and RNA. Proteases released during this process must be inhibited to prevent
degradation of the target recombinant protein.49-50,51
Separation of vlmH from E. coli native proteins is accomplished by affinity
chromatography. Figure 16 shows the interaction of the His6 tag (present in most of our
constructs) with a Ni2+ cross-linked resin. Affinity purification exploits specific binding
interactions such as this one to separate molecules. Ni2+ resin is immobilized to a solid
support and as cell lysate is applied, proteins with an affinity to the Ni2+ resin become
bound. Cell lysate proteins that do not interact are washed away using an appropriate
buffer that maintains the interaction between the bound protein and resin. The bound
proteins are separated from the solid support using a buffer that contains a competitive
ligand, imidazole in the case of Ni2+ affinity purification.52,53
25
MBP
vlmH
Figure 16: Illustration of the N-terminal tag interacting with Ni2+ resin. This is an
example of the fusion protein, His6-MBP-vlmH. The lone pair of electrons from the
nitrogen of the imidazole group interacts with the Ni2+ cross-linked resin.
Last, proteolysis can be used to remove unwanted tags or fusion proteins. These
tags or proteins can interfere with the activity or crystallization of the protein, so best
practice calls for their removal. Our vectors contain cleavable tags; that is, the tag is
attached to the protein through a linker sequence that has a unique cleavage site for a
specific protease. For example, the pLM302 vector’s MBP tag can be cleaved specifically
from the fusion protein using the PreScission protease. After incubation of the purified
protein with the protease, the tag and protease must be separated from the protein of
interest. In our case, nickel or cobalt affinity purification is used for this separation.
Materials and Methods
Materials
The codon-optimized vlmH gene was obtained from GenScript USA Inc,
Piscataway, NJ. Target vectors for subcloning include: pBG100, pET21b, pET24 (+),
pLM302 (obtained from the Vanderbilt University Center for Structural Biology), pGEXKG (from Paula Fischhaber, CSUN) and pET-SUMO (from Karin Crowhurst, CSUN).
Protein and DNA electrophoresis materials were from Bio-Rad Laboratories. The Agilent
26
Cary 60 UV-Vis Spectrophotometer was used for measuring the absorbance of bacterial
culture. The ÄKTA PrimePlus FPLC system and all protein purification columns and
resins were purchased from General Electric.
DNA Handling and Molecular Biology
E. coli Transformation by Heat Shock
DH5α E. coli cells were used for plasmid propagation and BL21 strains were used
for protein expression. To transform cells, plasmid DNA and competent cells were
incubated at 0 °C for 10 - 30 minutes. The mixture was heat shocked for 5 minutes at
37 °C to allow entry of DNA into cells and then allowed to rest for 2 minutes at 0 °C.
Cells were incubated with shaking in media at 37 °C for 1 hour to allow expression of the
vector’s antibiotic resistance gene. This recovery period is followed by selection on LBagarose plates supplemented with the appropriate antibiotic (Kanamycin 50 µg/mL or
Ampicillin 100 µg/mL). Plates containing colonies were stored at 4 °C.
Purification of Plasmid DNA
Under sterile conditions, a transformed colony was selected and used to inoculate
5 mL of LB media with appropriate antibiotic. The cell culture was shaken at 225 rpm at
37 °C overnight. After the overnight growth, the plasmid was isolated from these cells
using the Qiagen Plasmid Miniprep Kit or Zyppy Plasmid Miniprep Kit.
Subcloning
The parent vector containing the vlmH gene (pUC57-vlmH) and the target
vector(s) were double digested with the restriction enzymes BamHI and XhoI for 45
minutes at 37 °C. The target vectors were also treated with alkaline phosphatase (AP) to
prevent self-ligation. The digested DNA samples were purified by DNA gel
27
electrophoresis and extracted from the agarose using the Zymoclean Gel DNA Recovery
Kit. The vector and vlmH DNA were ligated following the manufacturer
recommendations by incubation with T4 DNA ligase and ATP at 4 °C overnight. The
ligation reactions were transformed into DH5α E. coli cells and plasmids were purified
from the resulting colonies using the methods described above.
Confirmation of Correct Gene Insertion
Ligation of vector and vlmH were confirmed by double digestion with BamHI
and XhoI. Plasmids were digested and separated by DNA gel electrophoresis. A
successful ligation is indicated by the presence of two bands, plasmid and vlmH gene at
the appropriate molecular weight. Successful ligations were sequenced at Laragen, Inc.
(Culver City, CA) to confirm proper insertion of the vlmH gene and to check for any
undesired mutations.
Expression of vlmH in E.coli
Under sterile conditions, a single colony containing vlmH DNA in an expression
strain is used to inoculate a 5 mL LB starter culture with appropriate antibiotic. The
inoculated culture is incubated with shaking at 37 °C for 12 - 16 hours. The overnight
inoculum is diluted 1:1000 into LB media with appropriate antibiotic. The optical density
(OD) of the culture is recorded by measuring the absorbance of the cell culture at 600 nm
(A600). When the OD reaches 0.6 absorbance units (Au), the log phase of growth, IPTG is
added to a final concentration of 0.5 mM.
The induction temperature and time varied depending on the experimental goals.
Reduced induction temperatures and times are noted in the text. At the end of the
induction period, the entire culture volume was harvested by centrifugation at 5,000 rpm
28
for 15 minutes. Harvested cell pellets were resuspended in 1mL lysis buffer per gram of
cell weight and stored at -20 °C for later use. Table 4 summarizes the lysis buffers used.
Table 4: Lysis buffers used to resuspend harvested cell pellets.
Lysis Buffer
20 mM Tris
200 mM NaCl
2 mM β-Mercaptoethanol (βME)
pH 8.0
50 mM NaH2PO4
150 mM NaCl
10 % Glycerol
1 mM DTT
pH 7.5
50 mM Tris
150 mM NaCl
30 % Glycerol
1 mM βME
0.5 mM EDTA
pH 7.5
Protein Type
His6- vlmH
vlmH
GST-vlmH
His6-MBP-vlmH
SUMO-vlmH
His6-MBP-vlmH
Tris buffers were used as a starting point for optimization of all buffers.
Cell culture samples for SDS-PAGE were collected and treated as follows. 100
L of culture is collected and the cells were harvested by centrifugation. The OD600 of
the culture measured is used to calculate the volume of loading buffer necessary to
normalize for the concentration of cells using the equation
#µL loading buffer = [(OD600 of your culture) ÷ 0.6] × 100 L
Protein Purification Methods
Cell Lysate Preparation
All steps in the purification process were performed at 4 °C. Cells in lysis buffer
were incubated on ice with PMSF (1 mM) and DNase/RNase (10 μg/mL). Lysozyme was
added to 0.25 mg/mL final concentration and cells were sonicated at 35 - 40 % amplitude
29
for 5 - 10 total minutes in 5 seconds on, 5 seconds off bursts to prevent sample
overheating. Lysed cells were centrifuged at 4 °C at 18,000 rpm for 30 minutes. The
soluble and insoluble components are separated and the supernatant is used for protein
purification. Samples of the supernatant and pellet were collected for SDS-PAGE
analysis.
Gravity Column Affinity Chromatography
Gravity flow chromatography was used for selected cell samples. The cell lysis
supernatant was added to 1 mL of Ni-NTA resin pre-equilibrated in equilibration buffer
(20 mM Tris, 200 mM NaCl, 2 mM BME, 2.5 mM imidazole, pH 8.0). The resin and
supernatant were incubated at 4 °C for 1 hour with gentle shaking to facilitate protein
binding to the resin. After incubation, the protein-resin slurry was centrifuged at 4 °C at
5,000 g for 15 minutes. The supernatant was removed and the resin washed in Buffer A
(20 mM Tris, 200 mM NaCl, 2 mM BME, 15 mM Imidazole, pH 8.0), and transferred to
a 10 mL plastic gravity-flow column. The column was washed with 1 column volume
(CV) of Buffer A. After washing, Buffer B (buffer A supplemented with 250 mM
imidazole) was applied to the column and ten 1 mL fractions were collected for SDSPAGE analysis.
FPLC Affinity Chromatography
The FPLC purification system was used for the majority of affinity
chromatography separation steps. The cell lysis supernatant is filtered using a 0.45 µm
filter and loaded manually into a sample loop for injection onto a Ni2+ charged column.
The elution of vlmH is monitored using a UV detector; increases in absorbance indicate
the presence of protein. As Buffer B is applied (Buffer A + 0.5 M imidazole), fractions
30
that correspond to increases in absorbance at 280 nm are collected and samples are saved
for SDS-PAGE analysis. Table 5 summarizes buffers used in the purification of His6MBP-vlmH.
Table 5: Buffers used to purify His6-MBP-vlmH.
Equilibration
Buffer A
His6-MBP-vlmH
50 mM NaH2PO4
150 mM NaCl
10 % Glycerol
1 mM DTT
pH 7.5
50 mM NaH2PO4,
150 mM NaCl
10 % Glycerol
1 mM DTT
pH 7.5
His6-MBP-vlmH
50 mM Tris
150 mM NaCl
30 % Glycerol
0.5 mM EDTA
1 mM βME
20 mM Imidazole
pH 7.5
50 mM Tris
150 mM NaCl
30 % Glycerol
0.5 mM EDTA
1 mM βME
20 mM Imidazole
pH 7.5
Buffer B
50 mM NaH2PO4
150 mM NaCl
10 % Glycerol
1 mM DTT
0.5 M Imidazole
pH 7.5
50 mM Tris
150 mM NaCl
30 % Glycerol
0.5 mM EDTA
1 mM βME
0.5 M Imidazole
pH 7.5
Phosphate is a very commonly used protein purification buffer. Tris buffer is used by
protein crystallographers to avoid false positive crystallization experiments due to
phosphate crystallization.
Proteolysis of His6-MBP-vlmH
The vlmH vector pLM302-vlmH expresses the fusion protein His6-MBP-vlmH.
The purification tag is no longer needed after purification. The PreScission Protease
targets the consensus sequence: LeuGluValLeuPheGlnGlyPro and cleaves in between
glutamine and glycine residues. Three variables were examined to determine the
appropriate conditions that resulted in complete cleavage of His6-MBP-vlmH: ratio of
protease to fusion protein, time, and temperature. Ratios of 1:50 and 4:50 protease to
fusion protein were tested to determine the best ratio that resulted in complete cleavage in
a short amount of time. Tag cleavage was carried out by incubation at 4 °C for 90
31
minutes (higher concentrations of proteins) or 12 - 16 hours in dialysis (lower protein
concentrations, immediately after recovery from the Ni-NTA column). The His6-MBP tag
is removed by a second Ni-NTA affinity purification step.
Results and Discussion
Construction of VlmH Vectors
The first three constructs, pBG100-vlmH, pET21b-vlmH, and pET24-vlmH were
constructed and verified by Dr. Vey. The three final vlmH expression vectors, pLM302vlmH, pGEX-KG-vlmH, and pET-SUMO-vlmH, were made and verified with help from
Nancy Huynh (CSUN undergraduate). These vectors express a protein tag at the Nterminal. Figure 17 illustrates the results of a representative series of double digestions.
The vlmH gene (~1150 bp band, lanes 2,3) was cut out of the pUC57 source plasmid
(visualized between the 2000 and 2500 bp markers). The target plasmids pLM302 (lanes
4&5) and pET-SUMO (lanes 6&7) were singly digested to verify that the restriction
enzymes were functional and digesting the vectors in only a single location, as expected.
vlmH
Figure 17: Double digestion of pUC57-vlmH, pLM302 and pETSUMO. All three
vectors were digested with BamHI and XhoI. Lanes are as follows: 1) 5000 bp DNA
ladder 2-3) pUC57-vlmH double digest 4) pLM302 BamHI digest 5) pLM302 XhoI
digest 6) pETSUMO BamHI digest 7) pETSUMO XhoI digest
32
After gel purification, ligation, transformation and purification of the ligated
vectors, each pure vector was doubly digested to confirm presence of the vlmH insert. A
representative successful ligation reaction (that of pGEX-KG and vlmH) is shown in
Figure 18.
vlmH
Figure 18: Double digestion of pGEX-KG-vlmH. Two colonies were cultured and each
vector purified. DNA gel electrophoresis verifies insertion of the vlmH gene by double
digestion. Lanes are as follows: 1) 5000 bp DNA ladder 2) Double digest of plasmid 1,
3) Double digest of plasmid 2
A single high intensity band (Figure 18, lanes 2-3) above the 5000 bp marker indicates
the presence of the plasmid. Despite the faint appearance of bands (lanes 2-3) at
approximately 1150 bp, the vlmH gene is present.
The ligation of vlmH into pET-SUMO required some optimization. An alternative
ligation strategy was used in addition to the one described above (Methods): a 1 hour,
22 °C incubation period followed by a 10 minute, 65 °C inactivation period.
33
vlmH
Figure 19: Digestion of pETSUMO-vlmH and pLM302-vlmH. Single and double
digestions were used to verify insertion of the vlmH gene. Lanes are as follows:
1) 5000 bp ladder 2) BamHI digest A 3) XhoI digest A 4) Double digest B 5)Double
digest B 6) BamHI digest B 7) XhoI digest B 8) Double digest 9) Double digest
10) BamHI digest 11) XhoI digest
Lane 4 confirms a successful ligation reaction using the alternative ligation method.
Unlike SUMO-vlmH, pLM302-vlmH did not require optimization (lanes 8-9). Lanes 2-3
and 6-7 in Figure 19 are single digestion controls for each empty target vector. These
samples were digested with each restriction enzyme to verify the restriction enzymes
were functional and digesting the vectors in only a single location, as expected.
The successful double digestions visualized by DNA electrophoresis tell us that
the vlmH gene of the correct length was inserted into our vector. Before continuing with
expression of vectors in E.coli, all six vlmH vectors were sequenced to check that vlmH
was inserted in the right direction, that nothing else was inserted, and that no mutations
were introduced.
34
Expression of VlmH Vectors in E. coli
Next, the vlmH vectors were subjected to expression trials in E. coli expression
strains to identify the best vector for vlmH production. Investigating which vector is
suitable is the first step of many to optimize expression in bacteria. Small scale (100 250 mL) expression is helpful to determine if soluble or insoluble vlmH is expressed and
the type of yields that could be obtained. If the vector yields soluble vlmH, the expression
is scaled up to (1 - 5 L) to obtain large quantities of vlmH to be purified. Overall, this is a
systematic process to insure the maximum amount of vlmH is produced.
pBG100-vlmH Expression in three E. coli strains
The vector pBG100-vlmH has a convenient yet simple His6-purification tag.
Expression of this vector in (DE3)-pLysS cells at 37 °C for 4 hours yielded a modest
amount of His6-vlmH (Figure 20 lane 4) but the protein appeared in the cell lysis pellet.
His6-vlmH
(40 kDa)
Figure 20: SDS-PAGE analysis of pBG100-vlmH expression in (DE3)-pLysS. Culture
induced at 37°C for 4 hours. Lanes are as follows: 1) Protein molecular weight standards
2) Control 3) Pre-induction 4) Post-induction 5) Cell lysis pellet 6) Cell lysis supernatant
35
In order to increase yields of soluble vlmH, the cell strain and the induction
temperatures were altered. The expression level of pBG100-vlmH was determined in two
additional strains, BL21 (DE3), and (DE3)-CodonPlus. As part of the modified
expression protocol the induction temperature was reduced in order to observe if reduced
temperatures facilitate proper protein folding. The expression trials of pBG100-vlmH in
BL21 (DE3) and (DE3)-CodonPlus are summarized in Appendix A. In summary, there
was no major improvement in vlmH expression in BL21 (DE3) and (DE3)-CodonPlus
even at reduced induction temperatures. A low yield of soluble vlmH was still observed.
Purification of pBG100-vlmH from pLysS cells induced at 37 °C was attempted (see
below).
pET24(+)-vlmH in E. coli BL21 (DE3)
Expression trials continued with the vector pET24(+)-vlmH. This vector was
constructed in our lab since it has been used previously by Parry and Li to express vlmH
with no purification tag.22 Even though the vector has been used to express untagged
vlmH, the expression protocol could require optimization to meet our goal of obtaining a
large quantity of vlmH. In our hands, pET24(+)-vlmH did not yield soluble vlmH (Figure
21 lanes 5,9,13).
36
vlmH
(40 kDa)
Figure 21: SDS-PAGE analysis comparing pET24(+)-vlmH expression in three
different E. coli strains. Culture induced at 37 °C for 4 hours. Lanes are as follows:
1) Protein standards 2) Pre-induction 3,6, 10) Post-induction 4) Cell lysis pellet 5) Cell
lysis supernatant 6) Pre-induction 7) Post-induction 8) Cell lysis pellet 9) Cell lysis
supernatant 10) Pre-induction 11) Post-induction 12) Cell lysis pellet 13) Cell lysis
supernatant.
VlmH is expressed at relatively low amounts in the post-induction, pellet and supernatant
samples in all three E. coli strains. Similar results were observed in the pET21b-vlmH
expression trials (see Appendix A). The vlmH band in the supernatant is faint and does
not indicate expression of highly soluble protein.
A summary of the pBG100-vlmH, pET21b-vlmH, and pET24(+)-vlmH
expressions trials are summarized in Table 6. Overall, the expression of vlmH in the
vectors pBG100, pET21b, and pET24 did not result in a high yield of soluble vlmH,
which is necessary for biochemical assays and crystallization experiments. Creating
fusion proteins between vlmH and proteins such as MBP, GST, or SUMO seemed
promising, given their success in improving solubility of other proteins.
37
Table 6: A summary of the initial vlmH constructs expressed in E.coli.
Expression
Vector
Purification
Tag
Cell Line(s)
Tested
pBG100-vlmH
His6-vlmH
BL21 (DE3),
CodonPlus,
pLysS
pET-21b -vlmH
pET-24(+)-vlmH
T7-vlmH
untagged
BL21 (DE3)
BL21 (DE3)
Temperatures
Tested
°C
37
30
25
18
37
37
Expression Level
Low in supernatant;
high in inclusion
bodies
Low
Low
pGEX-KG-vlmH, pLM302-vlmH, SUMO-vlmH Expression in E. coli BL21
(DE3)
The fusion proteins were subjected to expression trials next. All three vectors
were induced at 37 °C for 4 hours. Insoluble vlmH resulted at this temperature, which
was addressed by reducing the induction temperature and adjusting the length of the
induction period (see Appendix B).
The lowest temperature, 18 °C for 24 hours, was selected for expression of all
three vectors because at a lower temperature inclusion bodies are less likely to form and
more soluble proteins are produced. All three of the fusion protein constructs yielded
some soluble protein. The MBP-vlmH fusion construct gave the highest levels of soluble
protein (see Appendix B). This fusion construct was pursued for further expression trials.
The vectors containing protein tags were only transformed into the BL21 (DE3)
E. coli strain because our first expression trials using all three strains showed no
difference between the cell lines’ expression levels or location. BL21 (DE3) was selected
since it was the most appropriate for vlmH.
38
Expression Trials of pLM302-vlmH in BL21 (DE3)
After the expression trials of all six vlmH constructs, the vector pLM302-vlmH
yielded soluble protein and was taken forward to scale up for purification trials. The
translated sequence in Figure 22 shows the entire fusion protein sequence including the
MBP protein tag.
MGSSHHHHHHGSSMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPD
KLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAV
RYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEP
YFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTD
YSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGI
NAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATME
NAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNN
NNLGIEENLEVLFQGPGSMRSLDAARDTCERLHPGLIKALEELPLLEREAEGSPVLD
IFRAHGGAGLLVPSAYGGHGADALDAVRVTRALGACSPSLAAAATMHNFTAAMLF
ALTDRVIPPTDEQKKLLARVAPEGMLLASGWAEGRTQQDILNPSVKATPVDDGFIL
NGSKKPCSLSRSMDILTASVILPDETGQQSLAVPLIMADSPGISVHPFWESPVLAGSQ
SNEVRLKDVHVPEKLIIRGTPDDPGRLDDLQTATFVWFELLITSAYVGAASALTELV
MERDRGSVTDRAALGIQLESAVGLTEGVARAVRDGVFGEEAVAAALTARFAVQKT
LAAISDQAIELLGGIAFIKSPELAYLSSALHPLAFHPPGRTSSSPHLVEYFSGGPLEI
Figure 22: Amino acid sequence for the fusion protein His6-MBP-vlmH.
Magenta = His6 tag. Green = MBP (44 kDa). Blue = vlmH (40 kDa). The PreScission
Protease cuts at the red QG, highlighted in yellow is the consensus sequence. The
cleaved construct will start “GPGSMRSL”. Predicted Molecular Weight = 84.8281 kDa.
The His6-MBP-vlmH construct was expressed at three temperatures: 37 °C for 4 hours,
25 °C for 8 hours, and 18 °C for 24 hours. The results of the trials are summarized in
Appendix B. At all three induction temperatures, His6-MBP-vlmH overexpressed in the
post-induction samples. However, soluble His6-MBP-vlmH was observed only at 25 °C
and 18 °C. At 25 °C for 8 hours, the maximum amount of protein per cell is observed.
Overall, the expression trials showed that two induction temperatures, 25 °C and 18 °C,
are acceptable temperatures to synthesize soluble His6-MBP-vlmH.
39
Protein Purification
Our next step was to purify vlmH. In affinity chromatography, we aim to recover
a majority of the vlmH available and obtain a high purity sample, which can be used for
biochemical assays and crystallization trials. Two types of purification strategies were
used. Small-scale cultures were purified to analyze the yield that would be obtained. As
soon as expression and purification methods were finalized, large cultures were prepared
to obtain large quantities of vlmH.
Purification of His6-vlmH
The vector pBG100-vlmH yielded some soluble His6-vlmH when expressed in
pLysS at 37 °C for 4 hours. A test purification was done to determine what kind of yield
of soluble protein would be obtained (Figure 23). Even though a majority of His6-vlmH
was in the cell lysis pellet, the volume of the cell lysis supernatant was large enough that
a useful amount of protein could have been present.
His6-vlmH (40 kDa)
Figure 23: SDS-PAGE analysis of pBG100-vlmH expression in pLysS and affinity
purification. Two pBG100-vlmH plasmids were used for expression trials in order to
confirm that there were no major plasmid-to-plasmid differences that impacted protein
expression. pBG100-vlmH plasmid 2 is shown. Lanes are as follows: 1) Protein
molecular weight standards 2) Control 3) Pre-induction 4) Post-induction 5) Cell lysis
pellet 6) Cell lysis supernatant 7) Flow through 8) Bind 9) Wash 10-12) Elutions
40
Figure 23 shows bands corresponding to His6-vlmH in the purification samples (lanes 911). However, a low amount is present since His6-vlmH is lost to the pellet and to the
flow through. His6-vlmH present in the flow through indicates a poor interaction of His6vlmH with the nickel resin. The tag may have been occluded, or the band observed in the
cell lysis supernatant may have actually been a different protein of molecular weight
similar to vlmH. The His6-MBP-vlmH fusion protein was pursued in the hopes that a
different construct would result in improved solubility and interaction with Ni2+ resin.
Purification of His6-MBP-vlmH
The results of our expression trials showed that the highest expression levels of
His6-MBP-vlmH were obtained from growths at 25 °C for 8 hours. This condition should
also give the highest yields of purified vlmH. However, His6-MBP-vlmH growths were
routinely carried out using induction temperatures of either 25 °C (8 hours) or 18 °C (24
hours) for convenience.
A representative trace showing the elution profile of His6-MBP-vlmH from a NiNTA column is shown in Figure 24. Representative fractions from the FPLC run shown
in Figure 24 were analyzed by SDS-PAGE to show presence of His6-MBP-vlmH (Figure
26). The optimal imidazole concentrations for vlmH elution were identified by gradient
elutions and refined by multistep elution strategies. Inclusion of 20 mM imidazole in the
wash buffers prevented binding of impurities without interfering with binding by His6MBP-vlmH. His6-MBP-vlmH elutes completely from the column in 260 mM imidazole,
though some His6-MBP-vlmH does elute at lower concentrations. In a typical
purification, all fractions containing His6-MBP-vlmH were combined after the Ni-NTA
column, concentrated, and used for the next purification steps. Talon resin (which uses
41
Co2+ rather than Ni2+) was also tested for the affinity purification during the protocol
optimization process. No major benefit was observed by using Talon resin, so the
optimized protocol uses Ni-NTA resin for all affinity purification steps
Figure 24: UV Trace of Ni2+ column purification. The blue line represents mAu200nm.
The green line indicates the concentration of Buffer B, which contains 500 mM
imidazole. The three visible peaks correspond to (1) the flow through, (2) a peak that
elutes at 30 mM imidazole (fractions 20-24), and (3) a peak that elutes at 260 mM
imidazole (fractions 26-32).
Proteolysis of His6-MBP-vlmH
The use of a His6-MBP fusion partner served two objectives: to enable the use of
affinity chromatography and to increase the solubility of vlmH. Once the fusion protein is
pure, His6-MBP is no longer needed since further experiments will focus on activity
assays and crystallization of vlmH only. Therefore, proteolysis of the protein tag and
removal by a second chromatography step is the last step in purification.
42
After His6-MBP-vlmH was retrieved from the first Ni-NTA column,
concentrated, and quantitated, the PreScission Protease was used to cleave the His6-MBP
tag from vlmH. The molar ratio of protease to fusion protein, the proteolysis temperature,
and the proteolysis incubation time and conditions (in dialysis vs. post-dialysis) were
optimized. The final protocol uses a molar ratio of 4:50 (protease to His6-MBP-vlmH)
and an incubation temperature of 4 °C. The proteolysis step was carried out in dialysis
overnight (12 - 16 hours). The cleaved His6-MBP is removed from vlmH by a second
affinity purification step carried out on the FPLC (Figure 25), in which free vlmH is
found in the flow through and the His6-MBP is trapped by the column (and eluted with
500 mM imidazole). Figure 26 illustrates the effectiveness of these two steps of the
purification protocol. Again, Talon and Ni-NTA resin were tested during the procedure
optimization process, and our optimized procedure uses Ni-NTA for both affinity steps.
Figure 25: UV Trace of Ni2+ column purification. The blue line represents mAu200nm.
The green line indicates the concentration of Buffer B, which contains 500 mM
imidazole. The two visible peaks correspond to (1) vlmH (2) His-MBP tag and impurities
that elute at 500 mM imidazole. All fractions were analyzed by SDS-PAGE in figure 26.
43
Figure 26: SDS-PAGE analysis of His6-MBP-vlmH purification.
Lanes are as follows: 1) Protein standards 2) Pre-Induction 3) Post-Induction 4) Cell lysis
supernatant 5) Cell lysis pellet 6) His6-MBP-vlmH 7) His6-MBP + vlmH 8) vlmH step 1
9-11) Elution step 1 12) Elution step 2 13) vlmH step 2
Comparison of the 18 °C and 25 °C His6-MBP-vlmH Growths
Predictions that protein induction at 25 °C would yield more purified protein were
borne out by our results. Cells induced at 25 °C yield on average 4.08 mg of vlmH per
liter culture (0.51 mg pure vlmH per gram of cells) while cells induced at 18 °C yield on
average 2.1 mg of vlmH per liter culture (0.35 mg pure vlmH per gram of cells).
Surprisingly, vlmH samples from 18 °C inductions appear to co-purify with
oxidized flavin. Concentrated His6-MBP-vlmH samples are slightly yellow in color
(Figure 27), and this yellow color is retained after removal of the MBP tag. A UV-Vis
absorbance spectrum of His6-MBP-vlmH (Figure 28) confirmed that this color is due to
the presence of bound oxidized flavin. Flavin was not added to the growth or the protein
sample during purification, so flavin produced by the E. coli host organism must have copurified with the enzyme. To our knowledge, this is the first example of a class D FMO
44
binding with such high affinity to the oxidized form of FMN. Co-purification of flavin
with vlmH is not observed when expression is induced at 25 °C.
Figure 28: Absorbance spectra of His6-MBP-vlmH and oxidized flavin. Top: After
the purification of His6-MBP-vlmH, fractions appeared yellow in color. Bottom: UVVisible spectra were collected from the samples pictured (left) and compared to that of
free flavin (right).
Summary of Optimized vlmH Expression and Purification Method
Our optimized vlmH expression and purification procedure is carried out as
follows. E. coli BL21(DE3) cells containing the pLM302-vlmH expression vector are
45
grown in LB liquid media at 37 °C and induced at 25 °C (for 8 hours) or 18 °C (24 hours)
with 1 mM IPTG at an OD of approximately 0.6. Cells are resuspended in lysis buffer (50
mM tris, 150 mM NaCl, 1 mM ME, 0.5 mM EDTA, 30 % glycerol, pH 7.5) with 10
μg/mL DNase, 10 μg/mL RNase and 1 mM PMSF and lysed by addition of lysozyme
(0.25 mg/mL) and sonication (5 - 10 min total disruption time). After clarification of the
crude lysate, the supernatant is fractionated by Ni-NTA affinity chromatography on an
FPLC with impurities eluting in Buffer A (50 mM tris, 150 mM NaCl, 1 mM ME, 30 %
glycerol, 20 mM imidazole pH 7.5) and His6-MBP-vlmH eluting at 260 mM imidazole.
After concentrating all of the purified His6-MBP-vlmH to 11.98 mg/mL, PreScission
protease is added at a 4:50 (protease : fusion protein) ratio, and the sample is dialyzed
overnight into buffer (50 mM tris, 150 mM NaCl, 1 mM ME, 30% glycerol, pH 7.5) to
proteolyze the sample and remove all imidazole. Last, the His6-MBP is removed by NiNTA affinity chromatography on an FPLC with vlmH coming through in the flow
through.
Conclusion
One of the goals of this research project is to express soluble vlmH for continued
experiments including biochemical assays and crystallization trials. In order to achieve
this goal, vlmH expression vectors were constructed and analyzed for their ability to
express soluble vlmH. An expression and purification method has been established based
on expression trials and numerous test purifications. These methods yield soluble vlmH
to be used for biochemical and structural characterization. Identification of an ideal
expression vector, induction conditions, and purification method for vlmH fulfills the
first goal aimed to establish methods to study vlmH in our lab.
46
CHAPTER 3
INITIATION OF BIOCHEMICAL AND CRYSTALLIZATION STUDIES OF
ISOBUTYALAMINE N-HYDROXYLASE (VLMH)
Introduction
With an expression and purification protocol in place, we can produce large
quantities (4.08 mg of vlmH per liter of culture) of vlmH. Next, we must confirm that our
vlmH is active and later crystallize it for X-ray crystallographic structure determination.
Showing that vlmH is active is an important step in preparing it for structure
determination, as it must be folded correctly to be active. To determine vlmH’s
enzymatic activity, we need a method to determine changes in substrate or product
concentration per minute. The methods we use here to determine the concentrations of
substrate and product - isobutylamine (IBA) and isobutylhydroxylamine (IBHA),
respectively - were adapted from methods established by Ronald Parry.20 Our activity
assays must contain all of the components required by vlmH for catalysis, as well. As a
two-component monooxygenase, vlmH requires reduced flavin (produced by including
flavin, flavin reductase and NADH in the assay mixture), molecular oxygen, the
substrate, and catalase (which breaks down any hydrogen peroxide formed by catalytic
uncoupling).
Crystallization of vlmH will help us understand protein function. Specifically,
structural models identify the active site, conformational changes during catalysis, and
residues near the site of catalysis. Such models can be used to identify which amino acids
are involved in catalysis. In future studies, the vlmH structure will be determined by Xray crystallography to further elucidate the mechanism of catalysis by vlmH.
47
This chapter describes optimization of methods to assay vlmH and attempts to
crystallize vlmH. The immediate goals were: 1) confirming flavin reductase supplies
reduced flavin to support catalysis, 2) showing that IBHA formation occurs when vlmH
is present, and 3) identifying conditions that result in the crystallization of vlmH.
Methods for Detecting VlmH Activity
Parry and Li previously studied the enzyme activity of vlmH from Streptomyces
viridifaciens. VlmH, as a Class D flavin monooxygenase, requires reduced flavin, which
can be generated by flavin reductase and a coenzyme such as NADH. Flavin
monooxygenases use reduced flavin to activate molecular oxygen for insertion into an
organic substrate. In the case of vlmH, IBA is hydroxylated to yield IBHA. Parry and
Li’s assay monitored IBHA formation and the consumption of IBA.
We intend to monitor the formation of IBHA, and to do so, we need a signal to
monitor. Neither IBA nor IBHA have a chromophore, so indirect methods must be used
to quantitate their concentrations. The two methods used by Ronald Parry in his
investigations were (1) a coupled spectrophotometric assay that takes advantage of a
specific redox reaction between the hydroxylamine of IBHA and iron (III) and (2) an
HPLC assay in which the primary amine of IBA or the secondary amine of IBHA are
derivatized with a fluorophore.
The coupled spectrophotometric assay uses a detection reagent that contains
iron(III) and 2,4,6,tripyridyl-s-triazine (TPTZ), a reagent commonly used for the specific
detection of iron(II).54,55 The complex formed between Fe(II) and TPTZ is an intense
violet color and absorbs visible light strongly at 594 nm. In our assay workup, IBHA
reduces iron (III) from the detection reagent to iron (II) (Figure 29). Each iron (II) ion
48
generated by the redox reaction with IBHA forms a complex with two TPTZ molecules,
forming the complex Fe(TPTZ)22+ (Figure 29) which absorbs light at 594 nm. This
complex can be easily quantitated using the absorption coefficient (Ɛ) = 2.12 mM-1 cm-1,2
Quantitating the Fe(TPTZ)22+ complex allows us to indirectly determine the number of
moles of IBHA, which should be equal to the number of moles of Fe(TPTZ)22+ in the
final solution.
Figure 29: Detection method used to determine the concentration of IBHA. By
monitoring the absorbance of the complex Fe(TPTZ)22+ at 594 nm, the amount of iron
(II) in solution is calculated using the absorption coefficient (Ɛ) = 2.12mM-1 cm-1.2 The
total amount of IBHA formed in the assay is calculated using the Beer-lambert law A =
Ɛcl.
Derivatization is a direct method that has been used to measure vlmH activity.
IBA and IBHA both contain derivatizable primary (in IBA) and secondary (in IBHA)
amines. In our experiments, the amines are derivatized with the fluorophore, 4-chloro-7nitrobenzo-2-oxa-1,2,-diazole chloride (NBD-Cl) (see Figure 30 for a generalized
derivatization reaction between NBD-Cl and a secondary amine).
49
+
Figure 30: Derivatization of a generalized secondary amine with NBD-Cl. The
substrate, IBA and product, IBHA can be derivatized by NBD-Cl.
After the enzyme assay has been derivatized, the solution solutes are separated by HPLC
using a reverse-phase column. According to Parry’s results, the IBHA derivative is best
monitored at an absorbance of 450 nm. The retention time of the IBHA derivative is
expected at 12 minutes.20 In this thesis, product detection by the TPTZ method is reported
and in the future the HPLC method will be used to confirm the activity of vlmH.
Parry and Li report the specific activity of vlmH from the native organism as
188.0 nmol IBHA/min-mg of IBHA produced per minute.20 We will compare this value
to our own preparations of vlmH expressed in E.coli. With methods in place, we will
pursue further kinetic experiments to determine which amino acids participate in
catalysis.
Methods for Crystallization Experiments
Identifying crystallization conditions for a protein is one challenge in protein
crystallography. A typical crystallization cocktail has three components: buffer, salt, and
precipitant. The buffer controls the pH of the crystallization solution, while the salt is
used to influence the solubility and stability of the protein, and the precipitant promotes
nucleation of protein crystals.
50
Since there are infinite ways to combine the numerous buffers, salts and
precipitants used for protein crystallization, a “sparse matrix” screen is used to identify
initial crystallization conditions. A sparse matrix screen contains a specific number of
previously successful crystallization solutions that sample crystallization space (all of the
possible combinations of common crystallization components) somewhat randomly.
Once initial crystallization conditions are identified by the sparse matrix screen, those
conditions are optimized to improve crystal size, morphology and diffraction.
Optimization of conditions typically includes incremental changes in buffer, salt,
precipitant, the concentration of those species, and the pH.
Hanging Drop Vapor Diffusion Method
The hanging drop vapor diffusion method is the most common crystallization
technique. This method uses a sealed system in which water diffuses from a lessconcentrated sample droplet to a more-concentrated reservoir solution (Figure 31).
Figure 31: The hanging drop method. The cover slide is used to seal a well with both
the sample drop (with protein) and a reservoir solution that does not contain protein. The
system is allowed to equilibrate over days, weeks, months, and sometimes years.
51
This vapor diffusion from the small (2 - 4 L) sample droplet, which contains the protein
of interest, slowly concentrates the protein. The environment in the droplet shifts from a
dilute state to supersaturation. When the droplet reaches supersaturation, nucleation
(Figure 32) and crystal growth can occur. 56
Figure 32: Phase diagram of protein crystallization. When crystallization wells are
first prepared, the droplet is in the undersaturated state. The goal is to reach
supersaturation where nucleation can take place while avoiding the precipitation zone.
From reference 57.
Figure 32 illustrates the narrow region between the nucleation and precipitation
zone. If the concentrations in the droplet surpass supersaturation, (as often occurs
experimentally), precipitation results.
Alternative Optimization Strategies: Additives and Crystallization Under Oil
Sometimes standard optimization does not improve the diffraction quality, crystal
size or morphology enough. In such cases, one can use alternative optimization strategies
like additives, seeding, and manipulation of the vapor diffusion rate.
Additives are small molecules (salts, amino acids, and other biomolecules) that
are added to a crystallization drop to help improve crystal size, morphology and
52
diffraction quality. A successful additive binds to the protein near a crystal lattice contact
(a point on the surface of a protein molecule) to alter the lattice contacts to give a betterordered crystal.
Sometimes nucleation can occur too fast, which prevents the growth of a single
large crystal. Microseeding can help address nucleation that is uncontrolled and occurring
too quickly, controlling growth to obtain fewer, larger crystals.
Manipulation of the vapor diffusion rate can slow down crystal growth, yielding
larger or better-ordered crystals, or speed up crystal growth in the case of slow-growing
crystals. To slow down vapor diffusion and crystal growth rates, oils can be layered on
top of the reservoir solution (see Figure 31).
Discriminating Between Protein and Salt Crystals
Several tests can be used to confirm that a crystal is protein and not salt. The best
way to show that a crystal is protein is by collecting diffraction data from it. Protein
diffraction images contain many reflections, whereas a salt crystal will give few
reflections under the same parameters. If an X-ray source isn’t readily available, other
tests can be used. Some of those tests are described here.
IzIt dye will stain protein crystals selectively. Protein crystals have empty spaces,
called solvent channels, due to asymmetrical protein packing. Dyes can easily diffuse into
these solvent channels and change the color of the crystal. A salt crystal, on the other
hand, will not absorb the dye as they pack closely together and have no extra space in
their lattices. The large solvent channels in protein crystals also cause the crystal to be
very fragile. Light pressure will cause protein crystals to shatter, but salt crystals require
more force (the “crush test”).
53
Finally, protein crystals can rotate plane-polarized light weakly, due to the chiral
center in each L-amino acid. This property results in protein crystals appearing weakly
birefringent when viewed between two polarizers. Salt crystals will be either very
strongly birefringent, or not birefringent at all.
Materials and Methods
Materials
The vlmH assay materials include: NAD(P)H:FMN oxidoreductase of
Photobacterium fischeri (Roche Diagnostics); catalase (MP Biomedicals); nicotinamide
adenine dinucleotide disodium salt (reduced form, NADH) (Calbiochem). All general
reagents were purchased from: Sigma Aldrich, Fischer Scientific, and Acros Organics.
An Agilient Cary 60 UV-Vis Spectrophotometer was used for the vlmH and flavin
reductase assays. All crystallization supplies were purchased from Hampton Research.
Assay Methods
Flavin Reductase Activity Assays
Flavin reductase was used to maintain a steady supply of reduced flavin in our
assays. Flavin reductase activity was confirmed by monitoring NADH and FMN
concentrations spectrophotometrically at 340 and 440 nm, respectively, over a 20 – 30
minute time period. Assays contained 100 mM bis-Tris pH 7.5, 100 mM NaCl, 10 µM
FMN, 0.001 U/µL NAD(P)H:FMN oxidoreductase and 4 mM NADH. Our assay method
uses a solution without FMN and NADH to blank the spectrophotometer.
VlmH Activity Assay
VlmH activity was determined by monitoring IBHA production using the coupled
spectrophotometric detection method.20 Assays contain: 100 mM bis-Tris·HCl buffer pH
54
7.5, 100 mM NaCl, 0.4 U/µL catalase, 10 µM FMN, 0.001 U/µL NAD(P)H:FMN
oxidoreductase, 20 – 80 M vlmH, 10 mM IBA and 4 mM NADH in a final volume of
200 μL. Assays were incubated in 1.7-mL microcentrifuge tubes at 30 °C for 30 minutes
and then quenched as described below.
Coupled Spectrophotometric Detection Method
After the 30 minute incubation period, assays are quenched by addition of 10 %
(w/v) trichloroacetic acid (TCA) to a final concentration of 5 % TCA, giving a total
volume of 600 μL. Precipitated protein was separated by centrifugation. To 400 μL of
soluble components, 1 mL of TPTZ detection reagent (4 M sodium acetate buffer, pH
5.9, 0.24 mM TPTZ, and 0.8 mM FeCl3) was added to give a final volume of 1.4 mL.
The suspension is incubated at room temperature for 30 minutes. The absorbance of the
Fe(TPTZ)22+ complex is determined at 594 nm. Assays are measured against a control
assay mixture without IBA. The concentration of Fe(TPTZ)22+ should equal the
concentration of IBHA. The extinction coefficient Ɛ = 2.12mM-1 cm-1 is used to calculate
the concentration of IBHA.20 The reaction rate is nmol IBHA/min is calculated from the
A594 nm and the extinction coefficient, taking into account the two dilutions described
above (dilution factor from quenching = 3, dilution factor from addition of detection
reagent = 3.5, resulting in a total dilution factor of 10.5). See Equation 1.
𝑛𝑚𝑜𝑙 𝐼𝐵𝐻𝐴
𝐴594
10.5 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟
1𝑀
109 𝑛𝑚𝑜𝑙
=
×
×
×
× 0.0002 𝐿 𝑎𝑠𝑠𝑎𝑦 𝑣𝑜𝑙𝑢𝑚𝑒
𝑚𝑖𝑛.
Ɛ(𝑚𝑀−1 · cm−1 )𝑙(𝑐𝑚)
30 𝑚𝑖𝑛. 𝑎𝑠𝑠𝑎𝑦 𝑡𝑖𝑚𝑒
1000𝑚𝑀
1 𝑚𝑜𝑙
Equation 1: Calculation to determine the rate using the TPTZ method.
55
Crystallization of VlmH
Broad Matrix and Optimization Screens
Hampton Research’s Index and Crystal Screen sparse matrix screens were used to
identify initial conditions for prep 1 crystals (prepared from cells induced at 18 °C for 24
hours) and prep 2 crystals (prepared from cells induced at 25 °C for 8 hours). The
hanging drop vapor diffusion method was used for both preps of crystals. 1 μL of protein
sample (5 mg/mL vlmH in 50 mM Tris, 150 mM NaCl, 30 % glycerol, 1 mM βmercaptoethanol, 0.5 mM EDTA pH 7.5) was mixed with 1 µL crystallization solution
and sealed over 750 µL of reservoir (crystallization) solution. Drops were observed daily
for one week, and weekly or monthly thereafter until the well dried completely.
Additive Screen
The Hampton Additive Screen was used to improve the shape and size of prep 1
crystals (prepared from cells induced at 18 °C for 24 hours). Droplets contained 1 µL
protein sample, 0.8 µL concentrated crystallization solution (all components were
included at 5/4 their original concentrations) and 0.2 µL additive. The drop was sealed
over 750 µL of the original crystallization solution condition.
Microseeding
VlmH crystals were harvested from the drop and pulverized to form seeds. The
seeds were diluted in crystallization solution at three dilutions (1,000x, 10,000x,
100,000x) and used for crystallization. Droplets contained 1 µL protein sample, 0.2 µL
seed solution, and 0.8 µL of concentrated reservoir solution (all components were
included at 5/4 their original concentrations). The drop was sealed over 750 µL of
concentrated reservoir solution.
56
Crystallization Under Oil
The crystallization drop was equilibrated against 750 µL of concentrated reservoir
solution that had a layer of paraffin or silicone oil (150 µL or 250 µL) applied on top.
Tests to Discriminate between Salt and Protein Crystals
IzIt dye: The well with the suspected crystals was opened to access the drop. IzIt
dye (0.4 μL) was added to the drop and the well was re-sealed. The dye was allowed to
diffuse into the crystal for 24 hours. Crystals were observed the following day.
Birefringence: Crystal birefringence was assessed by viewing the crystals in
question between two polarizers. The base window was replaced with a polarized
window, and a second polarizer was attached to the microscope’s objective lens. Crystals
were viewed as usual.
Protein gel electrophoresis: Harvested crystals were washed twice in
crystallization solution and then dissolved in water. Samples were treated with protein gel
loading buffer and run on a protein gel. In order to see vlmH at the appropriate molecular
weight, a significant amount of crystals are required. In addition, large singular crystals
are best in order to avoid collecting precipitation. If precipitation is introduced, then this
will obscure the results of protein gel electrophoresis.
X-ray diffraction: Crystals were screened on Beamline 12-2 of the Stanford
Synchrotron Radiation Laboratory (Stanford Linear Accelerator Center, Menlo Park, CA)
via remote access. It is best to use this test when large three-dimensional crystals are
obtained since it will occupy the entire space of the loop. Crystals that are not large
enough can potentially result in poor quality data.
57
Results and Discussion
Activity Assays
The hydroxylation reaction of IBA to IBHA depends on the activity of two
enzymes: flavin reductase supplies reduced flavin to the vlmH, which can then catalyze
the hydroxylation. Flavin reductase was assayed to show that at the conditions used in
our vlmH assays, a sufficient amount of reduced flavin was available for vlmH catalysis
during the time period of the assay.
Flavin Reductase Provides Sufficient Reduced Flavin to Support vlmH
Catalysis
Assay mixtures that excluded vlmH and IBA were monitored
spectrophotometrically for 30 minutes to observe changes in the NADH and FMN
concentrations. At the concentrations used (4 mM NADH), the absorbance at 340 nm due
to the NADH was higher than 4.0 Au, and could not be measured accurately. However,
our results show that NADH does persist in solution throughout the 30 minute time
period (Figure 33), indicating that the supply of reducing equivalents was not exhausted.
The presence of oxidized flavin was monitored at 440nm. At the beginning of the assay
time period, 10 M FMN gave an absorbance at 440 nm of approximately 0.14 Au. That
absorbance disappeared within three minutes of the start of incubation, indicating that the
flavin was completely reduced (Figure 33). The flavin was maintained in its reduced form
throughout the 30 minute time period in the absence of vlmH and substrate. Notably,
NADH concentrations of less than ~0.6 mM were not enough to maintain flavin in its
reduced form due to nonenzymatic cycling of the flavin cofactor by dissolved O2 (data
not shown). These results confirm that under our experimental conditions, inclusion of
58
flavin reductase and NADH is a dependable method for generating reduced flavin over
the time period required by our assays (30 minutes).
Figure 33: FMN quickly reduces to FMNH2 and is maintained in its reduced form.
Reduced NADH is detected to perform the redox reaction with flavin reductase. In less
than 5 minutes, FMN was reduced to FMNH2. FMNH2 is present in its reduced form for
at least 30 minutes.
Addition of vlmH to a flavin reductase assay resulted in brief reappearance of the
A440 absorbance, indicating re-oxidation of flavin (Figure 34). This may have been
caused by the additional O2 dissolved in the vlmH sample, or nonproductive flavin
cycling by vlmH. Similar behavior was observed when substrate alone (no vlmH) was
added to the solution (data not shown). When both vlmH and IBA were added to a flavin
reductase assay, the A440 absorbance immediately increased and persisted (Figure 35),
again indicating reoxidation of the flavin. Due to the presence of substrate in this reaction
and by comparing these results to those in Figure 34, we tentatively took this to indicate
59
turnover by vlmH, though it could also be attributed to experimental error or
contamination with an oxidant.
FMNox. (vlmH only)
0.15
Au 440 nm
0.10
0.05
0.00
0
20
-0.05
40
60
80
Time (min)
Figure 34: Flavin reductase assay with the addition of vlmH and no substrate
present. VlmH (272 µg) was added at 30 minutes in the absence of the substrate IBA;
this assay also resulted in the immediate re-oxidation of flavin.
FMNox. (10 µM)
Au 440 nm
0.20
0.15
0.10
0.05
0.00
0
20
40
Time (min)
60
80
Figure 35: Flavin reductase assay with the addition of vlmH and substrate present.
VlmH (272 µg) and the substrate, IBA (10 mM) were added at 30 minutes. The addition
of both resulted in the immediate re-oxidation of flavin.
60
VlmH Catalyzes Formation of Isobutylhydroxylamine (IBHA)
The coupled spectrophotometric assay was used to determine more conclusively
whether our preparations of vlmH were catalytically active by measuring product
formation. Table 7 summarizes the results of selected individual assays, which were
carried out in buffers containing atmospheric levels of oxygen (20 %) or with pure
oxygen (100 %) incorporated by bubbling with 100 % O2.
Table 7: Raw data and calculated results from selected vlmH assays.
vlmH
O2
Avg. A594
Rate
(μM)
(%)
(Au)
(nmol/min)
0
20
0.0299 ± 0.0157 (3)
0.9873
0
100
1.473
0.0446 ± 0.0407 (2)
20
20
0.1549 ± 0.0456 (2)
5.115
20
100
0.1430
4.722
40
20
0.3204 ± 0.0827 (3)
10.58
40
100
0.4342
14.34
50
100
0.5633
18.60
The coupled spectroscopic assay was used to quantitate product formation. Standard
deviations are provided when more than one data point was collected, and the number of
repetitions of that point is given in parenthesis.
Our preliminary activity assays have shown that an increase in the rate is observed when
increased amounts of O2 are dissolved in the assay solutions (Table 7). We have also
shown that the rate of product formation (nmol/min) by vlmH increases with increasing
amounts of enzyme (Figure 36).
61
Rate (nmol/min)
Reaction Rate Increases with vlmH Concentration
20.0
18.0
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
R² = 0.9557
0
10
20
30
vlmH (µM)
40
50
60
Figure 36: Reaction rate increases with vlmH concentration. Data from experiments
including 100% O2 in the assays buffer are plotted here.
We would expect a linear increase in the amount of product formed when 100 % O2 is
present. Our results do show a linear increase (R2 = 0.95567). Taken together, our results
indicate that vlmH is active, and is likely using O2 to insert a hydroxyl group into its
substrate.
VlmH Crystallization
Two vlmH preps were used for initial crystallization experiments: “Prep 1”
(prepared from cells induced at 18 °C for 24 hours) and “Prep 2” (prepared from cells
induced at 25 °C for 8 hours). Differences in crystallization preferences were observed,
possibly due to presence of bound oxidized flavin in the Prep 1 sample (see Chapter 2).
62
Prep 1 Crystals
The Initial Crystallization Hit
Prep 1 protein was subjected to sparse matrix screening using the Hampton Index
and Crystal Screens. These experiments yielded one hit, which appeared as small needles
in the original crystallization solution (Figure 37). That hit had crystallization
components 0.1 M sodium cacodylate pH 6.5, 0.2 M magnesium acetate, and 20 % w/v
polyethylene glycol (PEG) 8,000.
Figure 37: VlmH needles obtained from Crystal Screen HR2-110. The needles were
grown in 0.2 M magnesium acetate tetrahydrate (Mg(CH3COO)2 • 4H2O), 0.1 M sodium
cacodylate trihydrate pH 6.5, and 20 % w/v PEG 8,000.
Standard Optimization by Simple Grid Screening
In order to improve the size and shape of the needles, the crystallization
components were systematically altered one by one to observe the effects on crystal
appearance and morphology. The specific components examined included: (1)
concentration of magnesium acetate, (2) pH of sodium cacodylate trihydrate, (3)
percentage of PEG 8,000, (4) identity of the precipitant. A representative optimization
63
screen is illustrated by Figure 38. At this point, the optimized crystallization solution was
0.1 M sodium cacodylate pH 6.5, 0.075 M magnesium acetate and 25 % w/v PEG 3350.
Mg(CH3COO)2 • 4H2O (M)
0.025
0.05
0.075
0.1
0.15
0.2
PEG 3350
20%
25%
30%
35%
Figure 38: Sample crystal optimization grid screen. Here, the percentage of PEG 3350
and Mg(CH3COO)2 • 4H2O were optimized. PEG 3350 concentration (% w/v) was
optimized in the vertical direction and magnesium acetate concentration was optimized in
the horizontal direction. All conditions contained 0.1 M sodium cacodylate pH 6.5. The
conditions identified as the most successful are indicated by the red box.
Additive Screening
Several additives were identified from the Hampton Additive Screen that
appeared to alter crystal morphology. Crystals grew in one to two days in the presence of
the additives shown Figure 39.
64
50 % v/v Polyethylene
glycol 400
40 % v/v Polypropylene
glycol P 400
0.1 M Betaine
hydrochloride
1.0 M NDSB-256
0.1 M Strontium
chloride hexahydrate
1.0 M Glycine
Figure 39: Compiled images and additive formulations from the Additive Screen
HR244. The drop outlined in red includes 50 % v/v PEG 400 as an additive. This
additive helped to stabilize the lattice, reducing precipitation and promoting the growth of
defined needles.
The crystals in Figure 39 all resemble different needle shapes with slightly different
macroscopic structure (they differ in length, width, and extent of branching). A
comparison of this drop with the original, shown in Figure 40, shows there is less
precipitation present, and the needles do not appear as thin. These additives appear to
help improve or alter crystal size.
65
Figure 40: Comparison of needles before and after additive screen. The crystals in
left drop have no additive while the crystals in the right drop contain the additive, 50%
v/v PEG 400. Both look “needle-like” however, there is less precipitation and fewer of
them in the right image.
Microseeding
Prep 1 crystals typically form quickly and in large numbers, with lots of
branching. We used microseeding to attempt to control the level of nucleation in each
drop, in the hopes of obtaining fewer but larger crystals. Microseeding at three seed
concentrations did appear to alter crystal nucleation (Figure 41), yielding crystals with an
improved shape (larger size and better separated). The results of microseeding are
summarized in Appendix D.
1,000x
10,000x
100,000x
Figure 41: Three different seed dilutions were used to initiate nucleation. Images are
compiled from row D4 from the microseed optimization screen, Appendix 1.
66
Crystallization Under Oil
As a final attempt to increase crystal size, we experimented with slowing the rate
of vapor diffusion by layering oil on the reservoir solution. A complete summary of the
results of crystallization under oil are summarized in Appendix D. Long, reasonably wellseparated needles appeared in conditions of higher percentages of PEG 3350 (25 %) with
250 µL of oil layered on the well. The size and shape appeared to improve with slower
vapor diffusion rates.
Tests to Confirm Protein Crystals
Three tests were used to determine whether the Prep1 crystals are protein or salt
(Figure 42). The blank drop test and crush test both suggested that the crystals are
protein, while the IzIt dye test contradicted that conclusions – either the crystals are salt,
or they are too small to see a color change due to the dye. The needles were not analyzed
using protein gel electrophoresis because there were not enough singular crystals to
collect from the drop. Large, singular crystals are preferred, while avoiding precipitation,
which will be detected on protein gel electrophoresis. In addition, the needles were not
screened for diffraction since they were small and thin. Attempts were made to harvest
them, however, multiple needles and precipitation were collected in the crystallization
loop, which will obscure data collection. More investigation of this crystal form is
necessary.
67
Figure 42: Results of three protein crystal tests. (a) Blank drop test: The drop on the
left shows typical crystallization results, while the drop on the right is a blank drop. (b)
IzIt dye: (left) Typical crystals incubated with IzIt dye for 24 hours (right) A lysozyme
crystal incubated with IzIt dye for 24 hours (example positive test result). (c) Crush test:
Typical crystals before (left) and after (right) crushing.
Prep 2 Crystals
Because Prep 1 crystals of vlmH seem to have oxidized flavin bound to vlmH, we
expect different conditions will be required to crystallize Prep 2 crystals of vlmH (8 hour,
25 °C induction period), which does not have oxidized flavin bound.
The Initial Crystallization Hit and Attempts to Reproduce
Small (<10 m) three-dimensional crystals were observed in 2 M NaCl and 10 %
PEG 6K (Figure 43). The conditions were optimized in order to increase crystal size.
68
Figure 43: VlmH crystals obtained from Crystal Screen HR2-110. The crystals were
observed in 12 days and grown in 2.0 M sodium chloride (NaCl) and 10 % w/v PEG
6,000.
The first optimization screens examined NaCl and PEG 6K concentrations, PEG
molecular weights, various cationic salts (NaCl, CaCl2, MgCl2 and LiCl), and altering
vlmH concentrations. Figure 44 documents the results from one screen that includes the
original conditions (2 M NaCl and PEG 6K). Our attempts to reproduce this hit failed.
0.5
NaCl (M)
1.5
2
1
2.5
3
PEG
Molecular Weight
2K
4K
6K
8K
Figure 44: Optimization screen of low to high molecular weight PEGs and NaCl
concentration. Crystals were grown in 10 % PEG for all precipitants and used a vlmH
concentration of 5 mg/mL. Outlined in red are the crystals grown using the initial
conditions.
69
Tests to Confirm Protein Crystals
Three tests were applied to determine whether the crystals are protein or salt. The
crystals were weakly birefringent (Figure 45) and a blank drop did not yield crystals.
Several crystals were screened for X-ray diffraction on Beamline 12-2 at SSRL, but they
did not diffract. This result may have been due to their small size.
Figure 45: Prep 2 crystal birefringence. The crystals are shown as viewed under (left)
normal conditions and (right) between two polarizers. Note the light colors (blue, pink,
yellow) observed in the image on the right.
Conclusion
We have established methods to generate and study the activity of purified vlmH.
Our results have shown that the flavin reductase and NADH used in our assays generate
sufficient amounts of reduced flavin to support vlmH activity under our experimental
conditions, and we have been able to detect product formation. We have also obtained
promising results that suggest our preparations of vlmH are active. Two crystal forms
were identified from initial crystallization screens, and progress was made toward
showing whether they are or are not protein, and optimizing those hits.
CONCLUSION AND FUTURE DIRECTIONS
We have successfully generated a reliable vlmH expression construct and have
established methods for purifying and studying vlmH. Six unique constructs were
70
generated and tested for stability and expression levels; of these, the His6-MBP-vlmH
construct had the highest expression levels and best solubility. This construct was
expressed on a large scale and purified. Optimization of the purification steps led to a
reliable method involving a metal affinity initial purification step, proteolysis with
PreScission protease, and a second metal affinity step to remove the protease, the cleaved
tag and any other impurities. A coupled spectrophotometric assay using Fe(TPTZ)22+ as a
chromophore was replicated in our lab and used to show product formation by our
purified vlmH samples. Finally, two crystal hits were identified, and efforts to (1)
confirm whether they are protein and (2) optimize them were initiated.
Future research on this project will draw upon the methods established here. First,
activity of vlmH will be explored through studies on how substrate concentration and pH
affect vlmH activity. The apparent binding affinity of vlmH for oxidized flavin will be
studied by spectrophotometric titration experiments using flavin-free preparations of the
enzyme. A second product detection method – derivatization and quantitation by HPLC –
will be used more extensively as well, to allow direct quantitation of smaller amounts of
product.
Two preliminary crystal forms have been identified. The behavior of unique vlmH
preparations in crystallization experiments does seem to vary, depending on the
temperature used during the induction period. FMN binding, which was observed
consistently upon induction at 18 °C (Prep 1), may be involved in these observed
differences. One crystal form (Prep 1) requires further optimization, and we were unable
to reproduce the other (Prep 2). In addition, our attempts to confirm whether these
crystals did indeed contain vlmH (and not salt) were inconclusive. First, purchasing
71
solutions directly from Hampton may help reproduction of the Prep 2 hit. Microseeding
could be helpful for this crystal form, as well. Next, any future Prep 1 optimizations
should include experiments at 4 °C to observe the effect of temperature on crystallization.
Finally, the glycerol content in the vlmH buffer (currently at 30 %) should be reduced, as
glycerol is known to interfere with crystallization experiments.
Last, the expression, purification and biochemical assay methods developed here
will be used to characterize vlmH mutants. The mutant forms of vlmH to be studied are
site-directed mutants of active site residues, and our goal in carrying out biochemical
experiments using these mutants is to understand their roles in catalysis. The methods
described here have already been applied to two vlmH mutants (S158A and H356A).
The results of this work and the research it has enabled will contribute to the
growing body of knowledge on the class D FMOs. Future biochemical assays will be able
to provide thorough kinetic data, which has not been reported for vlmH. For example, by
determining the dissociation constant (Km) we can measure the affinity of vlmH for a
ligand. A complete set of kinetic data could be helpful in understanding other class D
FMOs. The continued pursuit of crystallization will lead to a structure of vlmH. From the
structure we will be able to predict which amino acids participate in catalysis.
Additionally, we will be able to study conformational changes once a ligand-bound
structure is determined.
These initial studies are part of a larger goal of the Vey lab to ultimately
characterize vlmH. With a complete understanding of the structure and function of vlmH,
plans of rational design or engineering can move forward.
72
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Appendix A
SUMMARY OF VLMH EXPRESSION TRIALS IN E. COLI
Chapter 2 describes the construction of six vlmH expression constructs and
summarizes expression trials of those constructs. This Appendix includes the details of
several expression trials omitted from Chapter 2 for brevity.
pBG100-vlmH Expression Trials at 37 °C
Two pBG100-vlmH plasmids were used for expression trials in order to confirm
that there were no major plasmid-to-plasmid differences that impacted protein expression
(notated as ‘vector 1 or 2 shown’). No significant differences were observed. Figure 46
shows the results of pBG100-vlmH plasmid 2 expression trials in pLysS at 37 °C. There
is expression of His6-vlmH but it appears primarily in the cell lysis pellet (lane 5, Figure
46).
Figure 46: SDS-PAGE analysis of pBG100-vlmH, vector 2, in pLysS. Culture induced
at 37 °C for 4 hours. Lanes are as follows: 1) Protein molecular weight standards 2)
Control (no induction) 3) Pre-induction 4) Post-induction 5) Cell lysis pellet 6) Cell lysis
supernatant
78
pBG100-vlmH expression trials in BL21 CodonPlus cells show expression of
His6-vlmH at low yields and in the cell lysis pellet (Lane 3, Figure 47).
Figure 47: SDS-PAGE analysis of pBG100-vlmH in CodonPlus. Culture induced with
IPTG at 37 °C for 4 hours. Gel A = Vector 1 Gel B = Vector 2
Lanes are as follows: 1) Protein standards 2) Pre-induction 3) Cell lysis pellet 4) Cell
lysis supernatant
BL21(DE3) cells also overexpressed His6-vlmH at higher levels than pLysS and
CodonPlus E. coli (Lane 8, Figure 48), but again, the protein showed up in the cell lysis
pellet (Lane 9, Figure 4).
Figure 48: SDS-PAGE of pBG100-vlmH expressed in BL21 (DE3). Culture induced at
37 °C for 4 hours. Gel A = Vector 1 Gel B = Vector 2
Lanes are as follows: 1) Protein molecular weight standards 2) Control, no induction 3)
Control, after 4 hours 4) Cell lysis pellet 5) Cell lysis supernatant 6) empty, no sample
loaded 7) Pre-induction 8) Post Induction 9) Cell lysis pellet 10) Cell lysis supernatant
79
pBG100-vlmH Expression in BL21 (DE3) at 18 °C
Next, pBG100-vlmH was overexpressed in BL21 (DE3) at a reduced induction
temperature (18 °C) to help improve protein solubility. A longer induction time (24
hours) was used accordingly, and samples along the time course were taken. Figure 49
compares the expression level of two pBG100-vlmH vectors. Lower levels of His6-vlmH
are expressed and little soluble protein is observed (Lane 9 in Figure 49).
Figure 49: SDS-PAGE analysis of pBG100-vlmH expressed in BL21 (DE3). Culture
induced at 18 °C for 24 hours. Gel A = Vector 1 Gel B = Vector 2
Lanes are as follows: 1) Protein molecular weight standards 2) Pre-induction 3-8) Postinduction time course 9) Cell lysis supernatant 10) Cell lysis pellet. 11-12) No sample
loaded.
80
pET21b-vlmH Expression in BL21 (DE3) at 37 °C
pET21b-vlmH encodes for an N-terminally T7-tagged vlmH expression construct.
T7 is an 11 amino acid affinity tag. We did not observe significant overexpression in any
of these strains. For example, by comparison of lanes 2 and 3 in Figure 50. T7-vlmH is
expressed in the post-induction, pellet and supernatant samples in all three E. coli strains.
Expression in the cell lysis supernatant appears low in all strains (Lanes 5, 9 and 13,
Figure 50).
Figure 50: SDS-PAGE analysis comparing pET21b-vlmH expression in three
different E. coli strains. Culture induced at 37 °C for 4 hours. Lanes are as follows:
1) Protein standards 2) Pre-induction 3)Post-induction 4) Cell lysis pellet 5) Cell lysis
supernatant 6) Pre-induction 7) Post-induction 8) Cell lysis pellet 9) Cell lysis
supernatant 10) Pre-induction 11) Post-induction 12) Cell lysis pellet 13) Cell lysis
supernatant
81
Appendix B
SUMMARY OF GST-vlmH, MBP-vlmH, SUMO-vlmH EXPRESSION IN E. COLI
BL21 (DE3) CELLS
This appendix provides the details of expression trials of. In this series of
experiments, the 18 °C induction temperature was used first because it was expected to
be the most successful.
GST-vlmH, MBP-vlmH, SUMO-vlmH Expression in BL21 (DE3) at 18 °C
With each new vector, control samples of growing cell culture (no induction)
were analyzed by SDS-PAGE to insure vlmH is not synthesized without IPTG. GSTvlmH is successfully overexpressed by BL21(DE3) cells (Figure 51). Expression appears
to be low, and although the bands corresponding to the cell lysis pellet and supernatant
appear to be very dilute (lanes 8 and 9, Figure 51B), there is not a significant amount of
protein visible in the cell lysis supernatant sample.
Figure 51: SDS-PAGE analysis of GST-vlmH expression in BL21 (DE3).
(A) Control samples (B) Induced samples. Cultures were induced at 18 °C for 24
hours. Lanes are as follows: 1) Protein molecular weight standards 2) Pre-induction 3-7)
Post-induction Time Course 8) Cell lysis pellet 9) Cell lysis supernatant 10) no sample
loaded. *Note: The sample for lane 6 in figures A and B was switched during loading.*
82
His6-MBP-vlmH expression is observed in BL21 (DE3) cells, at low levels (Lane 9,
Figure 51B). See below for further experiments.
Figure 52: SDS-PAGE analysis of His6-MBP-vlmH expression in BL21 (DE3).
Control samples (A) Induced samples (B) Cultures induced at 18 °C for 24 hours.
Lanes are as follows: 1) Protein molecular weight standards 2) Pre-induction 3-7) Postinduction Time Course 8) Cell lysis pellet 9) Cell lysis supernatant 10) no sample loaded
SUMO-vlmH is also expressed in BL21(DE3) cells at reasonable levels (lanes 3-7,
Figure 53B), though expression is low in the supernatant and pellet samples (lanes 8 and
9, Figure 53B).
Figure 53: SDS-PAGE analysis of SUMO-vlmH expression in BL21 (DE3).
Control samples (A) Induced samples (B) Cultures induced at 18 °C for 24 hours.
Lanes are as follows: 1) Protein molecular weight standards 2) Pre-induction 3-7) Postinduction Time Course 8) Cell lysis pellet 9) Cell lysis supernatant 10) no sample loaded
83
All three fusion constructs are expressed at the appropriate molecular weight. The
maximum amount of vlmH produced is observed at 4 hours and is maintained throughout
the induction period. Among the three fusion constructs, the expression of His6-MBPvlmH in BL21 (DE3) showed best production of soluble protein. This result prompted
further expression trials at other induction temperatures.
His6-MBP-vlmH Expression in BL21 (DE3) 37 °C
Thorough expression trials of the construct pLM302-vlmH began with the
induction temperature 37 °C for 4 hours. His6-MBP-vlmH was expressed at high levels,
but appeared solely in inclusion bodies (in the cell lysis pellet, lanes 7 and 13, Figure 54).
Figure 54: SDS-PAGE analysis of His6-MBP-vlmH expression in BL21 (DE3).
Cultures induced at 37 °C for 4 hours. Lanes are as follows: 1) Protein molecular weight
standards 2) Pre-Induction 3-5) Post-induction time 6) Cell lysis supernatant 7) Cell lysis
pellet 8) Protein standards 9-11) Post-induction time course 12) Cell lysis supernatant 13)
Cell lysis pellet
84
His6-MBP-vlmH Expression in BL21 (DE3) 25 °C
Expression trials of the construct pLM302-vlmH were carried out next at 25 °C.
At this temperature, soluble His6-MBP-vlmH is clearly observed (Lanes 7 and 13, Figure
55) though some of the protein remained in the pellet (Lanes 8 and 14, Figure 55).
Figure 55: SDS-PAGE analysis of His6-MBP-vlmH expression in BL21 (DE3).
Cultures induced at 25 °C for 8 hours. Lanes are as follows: 1) Protein molecular weight
standards 2) Pre-Induction 3-6) Post-induction time course 7) Cell lysis supernatant 8)
Cell lysis pellet 9-12) Post-induction time course 13) Cell lysis supernatant 14) Cell lysis
pellet
85
His6-MBP-vlmH Expression in BL21 (DE3) 18 °C
Last, His6-MBP-vlmH was expressed at 18 °C for 24 hours. Soluble His6-MBPvlmH is observed again (Lane 8, Figure 56) and little appeared in the pellet (Lane 9,
Figure 56). Lower expression levels of the construct are observed at this lower
temperature, as expected.
Figure 56: SDS-PAGE analysis of His6-MBP-vlmH expression in BL21 (DE3).
Cultures induced at 18 °C for 24 hours. Lanes are as follows: 1) Protein molecular weight
standards 2) Pre-Induction 3-7) Post Induction time course 8) Cell lysis supernatant 9)
Cell lysis pellet
The expression trials of His6-MBP-vlmH identified two induction conditions to
obtain soluble vlmH, 25 °C or 18 °C with 0.5 mM IPTG used for induction (use of more
1.0mM IPTG had no effect on expression levels).
86
Appendix C
SUMMARY OF His6-vlmH PURIFICATION
pBG100-vlmH Expression in pLysS and CodonPlus at 37 °C
Expression of pBG100-vlmH in pLysS and CodonPlus at 37 °C gave some
soluble His6-vlmH, though most was insoluble. A small-scale purification was performed
to determine whether any purified protein could be isolated from these cells. The cell
lysis supernatant was incubated with NiNTA resin and a typical gravity column was run.
His6-vlmH did not bind to the nickel resin (lanes 7-10, Figure 57). This may be because
the tag was occluded. Very low yields of protein were obtained.
Figure 57: SDS-PAGE analysis of pBG100-vlmH expression in pLysS and affinity
purification. Two pBG100-vlmH plasmids were used for expression trials in order to
confirm that there were no major plasmid-to-plasmid differences that impacted protein
expression. Expression of vector 2 is shown. Lanes are as follows: 1) Protein molecular
weight standards 2) Control 3) Pre-induction 4) Post-induction 5) Cell lysis pellet 6) Cell
lysis supernatant 7) Flow through 8) Bind 9) Wash 10-12) Elutions
87
Appendix D
SUMMARY OF PREP 1 VLMH CRYSTALLIZATION EXPERIMENTS
Optimization of Prep 1 Crystals
The pH of the cacodylate buffer was optimized against the salt concentration.
This experiment confirmed that pH <6.5 did not yield crystals (Figure 58). Higher pH
values were not examined.
Magnesium acetate tetrahydrate (M)
0.025
0.05
0.075
0.1
0.15
0.2
pH 5.0
pH 5.5
pH 6.0
pH 6.5
Figure 58: Optimization of 0.1 M sodium cacodylate trihydrate pH to determine the ideal
pH for vlmH crystals. All drops contained 25 % w/v PEG 3350. Outlined in red are ideal
magnesium acetate tetrahydrate conditions that resulted in needles at a pH of 6.5.
Microseeding
Microseeding helped increase Prep 1 crystal size. The following images are drops
from one microseeding experiment and are organized by row from a 24-well crystal tray.
From rows A-D, [PEG] increased from 10 to 25 %. At higher PEG concentrations, more
crystals were observed. The effect of microseeding is clearly visible: as the concentration
of the seeds decreased, the number of crystals observed also decreased, as expected.
88
Magnesium acetate tetrahydrate
(M)
0.025
A1
A2
0.05
0.075
A3
0.1
A4
A5
0.15
A6
0.2
1,000x
10,000x
100,000x
Figure 59: Compiled images from Row A. Crystals grown in 0.1 M sodium cacodylate
trihydrate pH 6.5 and 10 % PEG 3350. Magnesium acetate tetrahydrate increased from
0.025 - 0.2 M. Dilutions of seeds incorporated into drops are indicated as 1,000x,
10,000x, and 100,000x.
89
Magnesium acetate tetrahydrate
(M)
0.025
B1
0.05
B2
0.075
B3
0.1
B4
B5
0.15
B6
0.2
1,000x
10,000x
100,000x
Figure 60: Compiled images from Row B. Crystals grown in, 0.1 M sodium cacodylate
trihydrate pH 6.5 and 15 % PEG 3350. Magnesium acetate tetrahydrate increased from
0.025 - 0.2 M. Dilutions of seeds incorporated into drops are indicated as 1,000x,
10,000x, and 100,000x.
90
Magnesium acetate tetrahydrate
(M)
C1
0.025
C2
0.05
C3
0.075
C4
0.1
C5
0.15
C6
0.2
1,000x
10,000x
100,000x
Figure 61: Compiled images from Row C. crystals grown in, 0.1 M sodium cacodylate
trihydrate pH 6.5 and 20 % PEG 3350. Magnesium acetate tetrahydrate increased from
0.025 - 0.2 M. Dilutions of seeds incorporated into drops are indicated.
91
Magnesium acetate tetrahydrate
(M)
0.025
D1
D2
0.05
D3
0.075
0.1
D4
0.15
D5
0.2
D6
1,000x
10,000x
100,000x
Figure 62: Compiled images from Row D. Crystals grown in, 0.1 M sodium cacodylate
tetrahydrate pH 6.5 and 25 % PEG 3350. Magnesium acetate tetrahydrate increased from
0.025 - 0.2 M. Dilutions of seeds incorporated into drops are indicated.
92
Crystallization Under Oil
Crystallization under oil also appeared to improve crystal separation and decrease
precipitation (Figure 61).
0.05 M magnesium acetate tetrahydrate
20 %
25 %
30 %
35%
Silicon
Paraffin
Combo
Figure 63: Compiled images of crystallization under oil. Crystals grown in 0.1 M
sodium cacodylate trihydrate pH 6.5 with a microseed solution of 100,000x. The PEG
percentage is given on the left of each row, and the salt concentrations are given at top of
each section. The type of oil layered in the well (150 L) is indicated at the bottom of
each column. Needles were observed in 1 to 2 days.
93
0.075 M magnesium acetate tetrahydrate
20 %
25 %
30 %
35 %
Silicon
Paraffin
Combo
Figure 64: Compiled images of crystallization under oil. Crystals grown in 0.1 M
sodium cacodylate trihydrate pH 6.5 with a microseed solution of 100,000x. The PEG
percentage is given on the left of each row, and the salt concentrations are given at top of
each section. The type of oil layered in the well (150 L) is indicated at the bottom of
each column. Needles were observed in 1 to 2 days.
94