Protein engineering projects - Dipartimento di Biotecnologie e

Protein engineering
projects
• Elena Rosini: “Novel, specific biosensors based on optimized glycine oxidase”
• Gianluca Conti: “Strategic manipulation of a cephalosporin C acylase”
• Paolo Motta: “Enzyme promiscuity of L-amino acid oxidases: a tool for
sustainable processes”
• Gianluca Molla: “SoAAO, characterization of a novel flavoprotein oxidase”
• Chiara Piatti: “Development of genetically encoded fluorescent sensors for
glycine and D-serine”
Lab meeting - 2014 february 12th
Varese
• Mattia Valentino: “Use of glycine oxidase for biotechnological applications:
production of enzyme variants”
1
Aim of the work:
M49S
H244K – M49L – A59R
His GO
Mutants
M49T
Grant support: No
Lab meeting - 2014 february 12th
Varese
H244K
M49L
2
Induction
• OD600 nm : 0.05
• At 37 °C
Start
• OD600 nm : 2 – 3
• 0.5 mM IPTG
• At 30 °C
• By Centrifugation
• After 24h
Collect
Growth in flasks (2L) with 500 mL of TB Broth
Lab meeting - 2014 february 12th
Varese
Methods:
3
Results:
Yield
mg/g
mg/L
H244K
13.6
0.8
10.9
H244K by Bioreactor
36.6
1.5
55.9
M49L
21.7
5.3
27.0
H244K – M49L – A59R
6.3
0.3
2.0
M49S
14.5
0.3
4.7
M49T
5.9
0.9
5.3
Future:
Use on Biosensor
Lab meeting - 2014 february 12th
Varese
Yield Growth
(g/L)
4
Aim: to produce an optimized enzyme to be employed in
specific biosensors
GLYCINE
(neurotransmitter in the CNS)
- Catatonia
- Ifosfamide encephalopathy
- Congenital nonketonic hyperglycemia
SARCOSINE
- Biomarker for prostate cancer in
biological fluids
Involved people: E. Rosini, L. Frattini, L. Piubelli, G. Molla, M. Valentino,
L. Pollegioni
Grant support: not
Lab meeting - 2014 february 12th
Varese
Novel, specific biosensors based on
optimized glycine oxidase
5
Novel, specific biosensors based on
optimized glycine oxidase
Methods:
 Site-saturation mutagenesis & screening
 Enzyme expression, purification and
kinetic characterization
 Site-directed mutagenesis
(introductionof multiple mutations)
Lab meeting - 2014 february 12th
Varese
 Molecular modeling
 Phylogenetic analysis
 Biosensor optimization
Collaborations: prof. S. D’Auria (Institute of Protein Biochemistry, CNR, Napoli)
6
Novel, specific biosensors based on
optimized glycine oxidase
Results:
Sarcosine
kcat,app (s-1)
Km,app (mM)
kcat,app/Km,app
kcat,app (s-1)
Km,app (mM)
kcat,app/Km,app
Wild-type
0.60 ± 0.03
0.70 ± 0.10
0.86
0.60 ± 0.02
0.70 ± 0.10
0.86
H244K
1.35 ± 0.05
0.14 ± 0.02
9.65
1.14 ± 0.05
0.78 ± 0.17
1.46
Glycine
Sarcosine
kcat,app (s-1)
Km,app (mM)
kcat,app/Km,app
kcat,app (s-1)
Km,app (mM) kcat,app/Km,app
Wild-type 0.60 ± 0.03
0.70 ± 0.10
0.86
0.60 ± 0.02
0.70 ± 0.10
0.86
Y246W
17.2 ± 3.40
0.07
0.87 ± 0.05
5.90 ± 0.90
0.15
1.21 ± 0.14
Glycine
Sarcosine
kcat,app (s-1)
Km,app (mM)
kcat,app/Km,app
kcat,app (s-1)
Km,app (mM)
kcat,app/Km,app
Wild-type
0.60 ± 0.03
0.70 ± 0.10
0.86
0.60 ± 0.02
0.70 ± 0.10
0.86
M49L
0.43 ± 0.04
1.35 ± 0.35
0.32
0.63 ± 0.03
0.12 ± 0.05
5.21
Glycine
Sarcosine
kcat,app (s-1)
Km,app (mM)
kcat,app/Km,app
kcat,app (s-1)
Km,app (mM)
kcat,app/Km,app
Wild-type 0.60 ± 0.03
0.70 ± 0.10
0.86
0.60 ± 0.02
0.70 ± 0.10
0.86
A54R
28.0 ± 3.00
0.04
1.30 ± 0.07
20.5 ± 2.21
0.06
Glycine
1.20 ± 0.10
Sarcosine
kcat,app (s-1)
Km,app (mM) kcat,app/Km,app
kcat,app (s-1)
Km,app (mM)
kcat,app/Km,app
Wild-type
0.60 ± 0.03
0.70 ± 0.10
0.86
0.60 ± 0.02
0.70 ± 0.10
0.86
M261R
1.00 ± 0.04
3.50 ± 0.30
0.30
0.90 ± 0.02
1.63 ± 0.15
0.53
Lab meeting - 2014 february 12th
Varese
Glycine
7
Novel, specific biosensors based on
optimized glycine oxidase
Results:
Specificity constant
Lab meeting - 2014 february 12th
Varese
Glycine
Sarcosine
8
Novel, specific biosensors based on
optimized glycine oxidase
Biosensor for glycine and sarcosine detection
Nile Red (1 µg/mL in ethanol)
GO (0.1 µM)
Lab meeting - 2014 february 12th
Varese
(Strianese M. et al., 2011)
Km (µM)
Wild-type
11 ± 0.7
H244K
1 ± 0.3
Glycine
Future: manuscript preparation
kcat,app (s-1)
Km,app (mM) kcat,app/Km,app
Wild-type
0.60 ± 0.03
0.70 ± 0.10
0.86
H244K
1.35 ± 0.05
0.14 ± 0.02
9.65
9
Strategic manipulation of a Cephalosporin C acylase
Lab meeting - 2014 february 12th
Varese
Aim: evolution of a CephC acylase for industrial applications by protein
engineering
Involved people:
Conti G., Rosini E., Molla G., Pollegioni L.
Grant support: not
10
Strategic manipulation of a Cephalosporin C acylase
D- amino acid
oxidase
CEPHALOSPORIN C
Spontaneously
Cephalosporin C
Acylase
Glutaryl-7-ACA
Acylase
7-ACA
Involved people:
Conti G., Rosini E., Molla G., Pollegioni L.
Grant support: not
Lab meeting - 2014 february 12th
Varese
Aim: evolution of a CephC acylase for industrial applications by protein
engineering
GLUTARYL-7-ACA
11
Strategic manipulation of a Cephalosporin C acylase
Methods:
Molecular
modeling
studies
INTEGRATED
APPROACH
Structure
Analysis*
Protein engineering
(Site-directed and site-saturation mutagenesis)
Protein expression and characterization
*Collaborations: Alice Vrielink – School of Chemistry and Biochemistry, University of Western Australia
Lab meeting - 2014 february 12th
Varese
Bioinformatics approaches
(Ancestral reconstruction)
12
Strategic manipulation of a Cephalosporin C acylase
Lab meeting - 2014 february 12th
Varese
Methods:
Protein engineering
(Site-directed and site-saturation mutagenesis)
Protein expression and characterization
13
Strategic manipulation of a Cephalosporin C acylase
Lab meeting - 2014 february 12th
Varese
Results:
14
15
Lab meeting - 2014 february 12th
Varese
Enzyme promiscuity of L-amino acid
oxidases: a tool for sustainable processes
To achieve this goal we intend to overexpress a recombinant form of
the L-amino acid oxidase from the actinomycetales Rhodococcus
opacus, using two different expression systems, Escherichia coli and
two species of the genus Streptomyces
Involved people: Loredano Pollegioni, Gianluca
Molla, Paolo Motta
Grant support: No
Lab meeting - 2014 february 12th
Varese
The aim of this project is the creation of an ‘’enzymatic tool’’ to be
employed in biotechnological processes on industrial scale, through
the exploitation of a family of enzymes called L-amino acid oxidases,
allowing us to also have an insight on the molecular basis that led to
the great differentiation observed in nature
16
Enzyme promiscuity of L-amino acid
oxidases: a tool for sustainable processes
Methods:
•
•
•
•
•
Molecular biology techniques: PCR, cloning, transformation, agarose gel
electrophoresis, intergeneric conjugation
Native PAGE, SDS-PAGE, western blot
FPLC, immobilized metal affinity chromatography
Spectrophotometric techniques
Enzyme activity assays: Peroxidase coupled assays (o-DNS, 4-amino
antipirine)
Collaborations:
Laboratorio di Biotecnologie Microbiche, Università dell’Insubria:
Flavia Marinelli, Francesca Berini
Lab meeting - 2014 february 12th
Varese
Heterologous proteinexpression :
17
Enzyme promiscuity of L-amino acid
oxidases: a tool for sustainable processes
Results:
Very high expression levels in E. coli  ≈ 100 mg per culture liter
Conditions: Induction= 0.1 mM IPTG, OD600 at induction = 0.6
100 % accumulates as inclusion bodies, can be solubilized using e.g. NLS or urea 
no activity
100
70
55
α-His
40
T0
3h
6h
Total cells
•
•
•
•
•
Inclusion
bodies
Crude
extract
70
55
40
Total cells
RoLAAO
Urea
RoLAAO
Total cells
NLS
Streptomyces, media tested: BTSB, MYM, SFM, V6, YEME (+/- MgCl2 and +/- sucrose), YEME 2X
One or two precultures
S. lividans: 2 clones with constitutive plasmid, 2 clones with inducible plasmid
S. venezuelae: 2 clones with constitutive plasmid
No expression
Lab meeting - 2014 february 12th
Varese
•
•
•
Future:
 Test inducible plasmid in S. venezuelae - Extracellular expression in E. coli
 Expression in Pichia pastoris (intracellular, peroxisomes, extracellular)
 Look for other potentially interesting L-amino acid oxidases
18
SoAAO, characterization of a novel flavoprotein oxidase
Aim:
Lab meeting - 2014 february 12th
Varese
Determination of the functional and structural features of the
aminoacetone oxidase from S. oligofermentas and understanding (?)
its physiological role.
Involved people: Gianluca Molla, Paolo Motta, Loredano Pollegioni
19
Grant support: none
SoAAO, characterization of a novel flavoprotein oxidase
Methods:
Spectroscopy (UV-Vis/CD)
Thermal stability
Enzymatic assays
FAD reactivity (midpoint redox potential, sulfite reactivity)
Size-exclusion chromatography
2. Determination of reaction products by mass spectrometry
3. 3D structure determination and analysis
Lab meeting - 2014 february 12th
Varese
1. Classical biochemical approach in flavinology
Collaborations:
Paola D’Arrigo - Dept. of Chemistry, Materials and Chemical Engineering - POLIMI
Marco Nardini, Martino Bolognesi - Structural Biology Laboratory - Dept. of
Biosciences - UNIMI
20
SoAAO, characterization of a novel flavoprotein oxidase
Results:
Very low activity -> ca. 45 mU/mg on aminoacetone
Structure:
New fold with 3 domains
(role of domain 3? Interaction with other proteins?)
No classical LAAO active site
Role: favor aminoacetone conversion to
methylglyoxal by Fe-containg enzymes?
Future: Definition of reaction products -> publication
Lab meeting - 2014 february 12th
Varese
Biochemistry: strong FAD binding
NO reaction with sulfite
Low midpoint redox pot. (-324 mV)
21
Aim: the aim of the project is the developing of
specific fluorescent indicators for the detection
of glycine and D-serine in the cells, exploitable
for the study of different diseases involving
alterated functionality of NMDA receptors.
Involved people: Chiara Piatti,
Caldinelli, Loredano Pollegioni
Laura
Lab meeting - 2014 february 12th
Varese
DEVELOPMENT OF GENETICALLY ENCODED
FLUORESCENT SENSORS FOR GLYCINE AND D-SERINE
22
Grant support: No
DEVELOPMENT OF GENETICALLY ENCODED
FLUORESCENT SENSORS FOR GLYCINE AND D-SERINE
Methods:
- Cloning of the synthetic gene in pET-24b(+).
- Expression tests for recombinant NR3B S1S2 segment
production in E. coli Origami2(DE3) strain.
T (°C) before
induction
37
O.D.
induction
2.5
[IPTG]
µM
30
T (°C) after
induction
17
Harvesting
O.N.
- Purification of NR3B S1S2 segment (AS precipitation, HiTrap,
GF).
Lab meeting - 2014 february 12th
Varese
- Design of the synthetic gene optimized for the expression of
NR3B S1S2 segment in E. coli cells.
23
DEVELOPMENT OF GENETICALLY ENCODED
FLUORESCENT SENSORS FOR GLYCINE AND D-SERINE
Lab meeting - 2014 february 12th
Varese
Results:
- Best medium: SB
- NR3B S1S2 precipitates at 50% AS
24
DEVELOPMENT OF GENETICALLY ENCODED
FLUORESCENT SENSORS FOR GLYCINE AND D-SERINE
μg
μg
Lab meeting - 2014 february 12th
Varese
- HiTrap  not pure
25
DEVELOPMENT OF GENETICALLY ENCODED
FLUORESCENT SENSORS FOR GLYCINE AND D-SERINE
μg
μg
Lab meeting - 2014 february 12th
Varese
- GF
26
Molecular basis of
diseases
• Pamela Cappelletti: “Nanostructured hydrogels for controlled release of
engineered therapeutic proteins against Parkinson’s disease-related
neurodegeneration”
• Pamela Cappelletti: “Biochemical characterization of human DAAO variants
involved in pathologies of central nervous system”
• Silvia Sacchi: “Structural/functional insight in human D-amino acid oxidasepLG72 interaction”
• Laura Caldinelli: “Expression and purification of the human recombinant
transcriptional factor PHOX2B”
• Luciano Piubelli: “Production of immunogenic proteins from Mycobacterium
tuberculosis for the design of a recombinant vaccine against tuberculosis”
Lab meeting - 2014 february 12th
Varese
• Luciano Piubelli: “Determination of D- and L-serine concentrations in plasma
as a diagnostic tool for neurodegenerative diseases”
27
Aim: evaluation of D- and L-serine concentrations
in human blood plasma of Alzheimer patients vs.
healthy people to develop a fast and cheap
diagnostic tool
Involved people: Luciano Piubelli, Loredano Pollegioni
Grant support: no
Lab meeting - 2014 february 12th
Varese
Determination of D- and L-serine concentrations in plasma
as a diagnostic tool for neurodegenerative diseases
28
Determination of D- and L-serine concentrations in plasma
as a diagnostic tool for neurodegenerative diseases
Methods: HPLC separation and quantification of D- and
Lab meeting - 2014 february 12th
Varese
L-serine upon derivatization with NAC and OPA
29
Collaborations: Prof. G. Bono, DBSV
Determination of D- and L-serine concentrations in plasma
as a diagnostic tool for neurodegenerative diseases
Lab meeting - 2014 february 12th
Varese
Results:
30
Future: analysis of further samples
The aim of the project is to obtain a nanostructured hydrogel based
system for drug controlled release based on an injectable resosrbable
hydrogel matrix and an engineered form of the chaperon protein
Hsp70 which prevents α-synuclein mediated neurotoxicity typical of
the Parkinson disease. This prototype has the promising capability to
reduce or stop PD symptoms and to be used as an innovative
therapeutic
device
for
PD
treatment.
In particular, the purpose of my work is the cloning, expression and
purification of Hsp70 (1A) and Tat-Hsp70 (1A) proteins.
Involved people: Pamela Cappelletti, Gianluca Molla and Loredano
Pollegioni
Grant support: Cariplo Biomateriali
Lab meeting - 2014 february 12th
Varese
Nanostructured hydrogels for controlled release of
engineered therapeutic proteins against Parkinson’s
disease-related neurodegeneration
31
Nanostructured hydrogels for controlled release of
engineered therapeutic proteins against Parkinson’s
disease-related neurodegeneration
Methods: Basic techniques of molecular biology; chromatographic
Collaborations:
• Politecnico di Milano: Carmen Giordano, Marta Tunesi;
• Istituto di ricerche farmacologiche M. Negri (MI): Diego
Albani, Serena Rodilossi;
• IMCB-CNR (NA): Luigi Ambrosio, Antonio Gloria, Teresa
Russo.
Lab meeting - 2014 february 12th
Varese
techniques; electrophoretic techniques; spectroscopic techniques; proteins
and enzymaticassays.
32
Nanostructured hydrogels for controlled release of
engineered therapeutic proteins against Parkinson’s
disease-related neurodegeneration





Purification of both Tat-Hsp70 (15 mg/L colture) and Hsp70 (30 mg/L
colture) with a degree of purity > 95%. Both proteinsare endo-free.
The Tat sequence doesn’t modify the secondary structure of Hsp70.
The Tat sequence doesn’t modify the ATPase activityof Hsp70.
We efficiently crossed-linked fluorescein to Hsp70 (NHS-Fluorescein
Antibody labeling kit, Pierce). The cross-linking of fluorescein doesn’t
alters the ATPase activity of Hsp70.
Finally, we demonstrated that Hsp70 interacts with an hydrogel matrix
constituted of PEG 2000 + collagen (1.8 mg/ml), while with a matrix of
collagen (1.8 mg/ml) alone, and using low quantity of Hsp70, there is
only a partial interaction.
Lab meeting - 2014 february 12th
Varese
Results:
33
Future: project deadline march 2015
Biochemical characterization of human DAAO variants
involved in pathologies of central nervous system
R199  W:
R199  Q:
W209  R:
mutation associated with familial ALS [Mitchell et
al., 2010];
mutation that affects the same amino acid found
mutated in some cases of ALS;
mutation localized on the dimerization surface of
hDAAO.
Involved people: Pamela Cappelletti; Andrea Cardone; Laura Caldinelli;
Lab meeting - 2014 february 12th
Varese
The aim of the project is the expression, purification and biochemical
characterization
of
the
following
hDAAO
variants:
Silvia Sacchi; Mattia Valentino; Luciano Piubelli and Loredano Pollegioni
34
Grant support: no
Biochemical characterization of human DAAO variants
involved in pathologies of central nervous system
Methods: Basic techniques of molecular biology; chromatographic
techniques; electrophoretic techniques; spectroscopic techniques; proteins
and enzymaticassays.
Results:
Purifications of hDAAO variants:
R199W
~ 3 mg/L colture
~ 90 % purity
~ 80 % apoprotein
R199Q
~ 2 mg/L colture
~ 90 % purity
~ 90 % apoprotein
W209R
~ 10 mg/L colture
> 95 % purity
100 % oloenzyme
Lab meeting - 2014 february 12th
Varese
Collaborations: none
35
Biochemical characterization of human DAAO variants
involved in pathologies of central nervous system
Enzyme
D-Serine
D-Alanine
FAD µM
Km (mM)
kcat (s-1)
Km (mM)
kcat (s-1)
Free form
Benzoate
complex
wt
5.9 ± 3.1
2.9 ± 0.1
1.1 ± 0.2
5.9 ± 0.2
7.9 ± 0.2
0.3 ± 0.1
R199W
>2000
>15
~ 440
~ 15
40.3 ± 2.6
9.5 ± 1.2
R199Q
>2000
>8
> 2000
> 10
33.5 ± 1.6 26.2 ± 0.4
1.2 ± 0.2
~ 118.7 ±
2.4
W209R
19.3 ± 3.5 71.8 ± 2.3
1.94 ± 0.1
0.15 ±
0.01
Lab meeting - 2014 february 12th
Varese
Kinetics properties and binding affinity for FAD of hDAAO variants
36
Biochemical characterization of human DAAO variants
involved in pathologies of central nervous system
Analysis of the oligomerization state of both holoenzyme and
apoprotein form of hDAAO variants and pLG72 binding
 Always dimeric oligomerizationstate
Interaction with pLG72
High concentration of the holoenzyme form  tetrameric
oligomerizationstate
R199W:
Low concentration of the holoenzyme and apoprotein
formform  dimeric oligomerizationstate
The elution volume of the tetramer is close to that of the
hDAAO-pLG72 complex
Lab meeting - 2014 february 12th
Varese
R199Q &
W209R:
37
Structural/functional insight in human
D-amino acid oxidase-pLG72 interaction
DAAO-pLG72 interaction as a way to define the
molecular mechanism by which pLG72 finely tunes
DAAO activity (and, consequently D-serine cellular
levels).
Involved people: Silvia Sacchi, Gianluca Molla,
Laura Caldinelli, Loredano Pollegioni.
Lab meeting - 2014 february 12th
Varese
Aim: the elucidation of the structural details of
38
Grant support:NO
Structural/functional insight in human
D-amino acid oxidase-pLG72 interaction
recombinant proteins expression and purification;
limited proteolysis and chemical cross-linking coupled to MS analysis;
molecular modeling;
site directed mutagenesis;
biochemical methods for the characterization of pLG72 and hDAAO
mutant variants  size exclusion chromatography
activity assay
spectrophotometric and spectrofluorimetric analyses
Lab meeting - 2014 february 12th
Varese
Methods:
Collaborations: Leila Birolo & Piero Pucci
Dipartimento Scienze Chimiche
Complesso Universitario Monte S. Angelo, Napoli
39
Structural/functional insight in human
D-amino acid oxidase-pLG72 interaction
Lab meeting - 2014 february 12th
Varese
Results:
40
Structural/functional insight in human
D-amino acid oxidase-pLG72 interaction
Lab meeting - 2014 february 12th
Varese
Results:
Future:
project deadline – summer 2014, publication
41
Expression and purification of the human
recombinant transcriptional factor PHOX2B
The isolation of pure recombinant PHOX2B will allow to start a deep
biochemical characterization of this transcriptional factor, with
specific focus on its 3D-structure determination.
This step is of the utmost importance to rationally design strategies
for the treatment of Congenital Central Hypoventilation
Syndrome(CCHS).
Involved people: Laura Caldinelli, Chiara Piatti and
Loredano Pollegioni
Grant support: NEPENTE project until April 2013, no grants at the
moment
Lab meeting - 2014 february 12th
Varese
The aim of this project is the production and the purification of
the recombinant human transcriptional factor PHOX2B in
Escherichia coli cells.
42
Expression and purification of the human
recombinant transcriptional factor PHOX2B
Collaborations: CNR- Institute of Neuroscience, Milan
Roberta Benfante, Simona Di Lascio
(TELETHON GRANT “New approaches to the molecular
pathogenesis of CCHS: implications for therapeutic
strategies”)
Lab meeting - 2014 february 12th
Varese
Methods: molecular biology, electrophoresis,
chromatography, spectroscopy, protein refolding
43
Expression and purification of the human
recombinant transcriptional factor PHOX2B
Results:
 on-column refolding,
 dilution in refolding buffer with additives (L-Arg or
detergents) followed by dialysis.
 HisPHOX2B refolded by dilution in refolding buffer with N-lauroylsarcosinebinds the specific
oligo in EMSA assay.
 Kd for protein-DNA complex determined by quenching of intrinsic Trp-fluorescence was in
micromolarrange.
 The protein is not stable: precipitation during ultrafiltrationor size-exclusion chromatography.
Future: 



protein refolding optimization (e.g. artificial chaperone-assisted refolding)
3D-structure determination
production and characterization of PHOX2B variants related to CCHS
production of PHOX2B variants with improved solubility
Lab meeting - 2014 february 12th
Varese
 Expression of PHOX2B recombinant protein in pET11a vector in BL21(DE3)pLysSE. coli cells.
 His-tagged protein purification under denaturing conditions by two chromatographic steps:
metal ion affinity chromatography (TALON) and size-exclusion chromatography (Superdex200
HiLoad): purity: 95%; yield: 2 mg/L of bacterial culture.
 Refolding experiments:  temperature-leap renaturation,
44
Production of immunogenic proteins from
Mycobacterium tuberculosis for the design of
a recombinant vaccine against tuberculosis
Lab meeting - 2014 february 12th
Varese
Aim: high-level heterologous production as soluble
proteins in E. coli of three antigenic proteins (TB10.4,
Ag85B, full) from M. tuberculosis
Involved people: Luciano Piubelli, Loredano Pollegioni
Grant support: Regione Lombardia, Banca del Monte di Lombardia
45
Production of immunogenic proteins from
Mycobacterium tuberculosis for the design of
a recombinant vaccine against tuberculosis
TB10.4, Ag85B
Production in E. coli BL21(DE3) using pET32b as fusion protein
with Trx and with His-tag
Purification by affinity chormatography
Cleavage with EK
TB10-4-Ag85B (full)
Production in E. coli BL21(DE3) using pColdI (full) with His-Tag
Purification by affinity chormatography
Collaborations:
Prof. Marinelli, Dr.ssa Binda (DBSV)
Univerisity of Pavia and Policlinico of Pavia
Lab meeting - 2014 february 12th
Varese
Methods:
46
Production of immunogenic proteins from
Mycobacterium tuberculosis for the design of
a recombinant vaccine against tuberculosis
TB10.4
Ag85B
full2
Protein glycosilation (Ara, Man, Ara-Man….) (UniPV)
Tests for biological activity (ELISA, ELISPOT) (Policl. PV)
Future: production of the proteins for glycosilation and
biological studies (Ag85B variants)
Lab meeting - 2014 february 12th
Varese
Results:
47
Biocatalysis & Biorefinery
• Mattia Valentino: “Production of L-aspartate oxidase for biocatalysis and
“system biocatalysis””
• Fabio Tonin: “Valor-Plus: development of an enzymatic tool box for lignin
degradation”
Lab meeting - 2014 february 12th
Varese
• Luciano Piubelli: “Production of aspartate ammonia lyase (aspartase) for use
in “system biocatalysis””
48
Aim of the work:
Biocatalysis Use
Immobilization
on resin
Collaborations: Prof. Servi’s Group (Politecnico di Milano)
Grant support: No
Lab meeting - 2014 february 12th
Varese
Production of St-LASPO
49
Methods:
• OD600 nm : 0.1
Start
• OD600 nm : 3 – 4
• 1 mM IPTG
• By Centrifugation
• After 24 h
Collect
Growth in flasks (2L) with 500 mL of SB or LB Broth,
always at 37 °C
Lab meeting - 2014 february 12th
Varese
Induction
50
LB Broth
SB Broth
5.1
11.8
mg/g
2.5
0.5
mg/L
13.2
5.8
U/g
0.8
0.2
U/L
4.0
2.5
Yield growth (g/L)
Yield
Future:
Optimization of storage conditions
Lab meeting - 2014 february 12th
Varese
Results:
51
Production of an aspartate ammonia lyase
(aspartase) for use in “system biocatalysis”
Involved people: Luciano Piubelli, Loredano Pollegioni
Lab meeting - 2014 february 12th
Varese
Aim: heterologous production in E. coli of a
thermostable aspartase from Bacillus
52
Grant support: no
Production of an aspartate ammonia lyase
(aspartase) for use in “system biocatalysis”
Methods:
Production in E. coli using pBAD
Purification by:
heat treatment
ammonium sulphate precipitation
IEC on Q-Sepharose
Lab meeting - 2014 february 12th
Varese
(induction using arabinose)
53
Collaborations: prof. Servi’s group
Production of an aspartate ammonia lyase
(aspartase) for use in “system biocatalysis”
Lab meeting - 2014 february 12th
Varese
Results:
Yield: 800 U/L (about 1 mg), recovery 50-60%
Future: optimization of the production and of the
purification procedure
54
Project Valor-Plus
Fabio Tonin, Elena Rosini
Supervisor: Loredano Pollegioni
Lab meeting - 2014 february 12th
Varese
Development of an “Enzymatic
Tool Box” for Lignin Degradation
55
Grant support: European Project
Project Valor-Plus
Lignin is an amorphouspolymerthat is foundin the cellwall of plant cells. The enzymatichydrolysisof ligninis a “green”alternative to chemical
degradationand allowthe use of the degradationproductsas starterin syntheticchemistryor the developmentof new products.
Identification of Enzymes that could play a role in
Lignin degradation
Laccase:
Heterologous Expression:
Laccase:
-Trametes versicolor
-Bacillus licheniformis (in E. coli)
-Pleurotus ostreatus
-Mycelioptera termophila (in S. cerevisiae)
-Rhus vernicifera
-Bjerkandera adusta (versatile)
Enzymes from
Actinomycetes
Fermentation Broth:
Lignin Peroxidase:
Manganese Peroxidase:
Manganese Peroxidase:
-Nematoloma frowardii
-Bjerkandera adusta
-Bjerkandera adusta (versatile)
-Nonomuraea sp. ATCC 39727 (in
collaboration with Flavia Marinelli and
Carmine Casciello)
Lab meeting - 2014 february 12th
Varese
Commercial Enzymes:
56
Collaborations: Servi & Turri Groups (Politecnico di Milano)
Project Valor-Plus
First of all: Set-up of Enzymatic Assays for Laccase, MnP and LiP
LACCASE
ABTS
Recombinant
2,6-DMP
kcat (s-1)
Km (μM)
kcat/Km
kcat (s-1)
Km (μM)
kcat/Km
kcat (s-1)
Km (μM)
kcat/Km
Trametes versicolor
18.6
31.5
0.6
4.8
410
0.01
12.2
16.2
0.8
Pleurotus ostreatus
11.0
28.3
0.4
3.7
770
0.01
3.9
27.2
0.2
Rhus vernicifera
2.3
Bacillus licheniformis
12.5
2600
0.002
1.9
14.2
0.9
0.1
3.9
1870
0.002
3.9
Lab meeting - 2014 february 12th
Varese
Commercial
Catechol
Laccase from Bacillus licheniformis 
Recombinant Expression into E. coli BL21 (DE3)
Yield: 20 mgprotein/Lcolture
Mn-PEROXIDASE
H2O2
Commercial
kcat (s-1)
Km (μM)
kcat/Km
Ki (mM)
Nematoloma frowardii
4.21
246
0.02
/
Bjerkandera adusta (versatile)
0.16
16
0.01
2.74
57
MM-L + Agar  47 U/Lbroth
Fermentation Broth of Nonomuraea
sp. ATCC 39727 had shown MnPeroxidase activity
mUtot iniziali
mUtot [(NH4)2SO4] 30%
mUtot [(NH4)2SO4] 50%
mUtot [(NH4)2SO4] 70%
MML-Agar
558
86
26
521
MML-YE
550
15
47
206
Future: (project deadline 4 years)
•Expression and characterization of Laccase from Mycelioptera termophila
Lab meeting - 2014 february 12th
Varese
MM-L + Yeast Extract  27 U/Lbroth
•Indentification of other interesting Lignolytic activities
•Test on Lignin Model Compound
•Characterization of Nonomuraea broth
58
Other project
Lab meeting - 2014 february 12th
Varese
• Elena Rosini: “Gene delivery of oxidative stress”
59
Gene Delivery of Oxidative Stress
2.5% O2
Lab meeting - 2014 february 12th
Varese
Aim: induction of cytotoxic oxidative stress by a
gene-directed enzyme prodrug therapy (GDEPT)
0.1 mM D-Ala
60
Involved people: E. Rosini, L. Pollegioni
Grant support: not
Gene Delivery of Oxidative Stress
Methods:
NO SKL
NO STOP
+ Kozak sequence
+ SacI/EcoRI
Kozak-DAAO-EYFP
 Cell transfection
Bioreducible polymer
HeLa (cervical cancer)
SKOV-3 (ovarian adenocarcinoma)
Lab meeting - 2014 february 12th
Varese
 Vectors preparation
61
Collaborations: dr. G. Candiani (Politecnico di Milano)
(Milane et al., 2011)
Gene Delivery of Oxidative Stress
Preliminary results:
1. Activity assay
EYFP
wt
R285A Q144R
Lab meeting - 2014 february 12th
Varese
Ctrl-
2. Enzyme expression
Cytotoxicity assay → op miza on
GSH quantification /(depletion)
Response to hypoxic stress
62
Future: search for financial support?
(Stegman et al., 1998)

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