EMBO Practical course on protein expression, purification and
crystallization’ – PEPC5, 2006
Expression of membrane proteins for
structure determination
Reinhard Grisshammer
NINDS, NIH, Bethesda MD, USA
Department of Health and Human Services
objectives
• expression of membrane proteins for structure
determination (not for ‘functional’ experiments)
– large amounts (milligram quantities)
– functional, correctly-folded membrane protein
– source: membrane
– alternative source: inclusion bodies → refolding to
obtain functional membrane protein
• bacterial vs. eukaryotic membrane proteins
topics
• general considerations, mechanism of membrane
insertion, topology
• examples of overexpression of bacterial and
eukaryotic membrane proteins in various hosts
systems
• functional expression of G-protein-coupled receptors
(GPCRs) in E. coli
• factors influencing the expression levels and stability
of GPCRs
• functional expression of GPCRs in eukaryotic hosts
topics
• general considerations, mechanism of membrane
insertion, topology
• examples of overexpression of bacterial and
eukaryotic membrane proteins in various hosts
systems
• functional expression of G-protein-coupled receptors
(GPCRs) in E. coli
• factors influencing the expression levels and stability
of GPCRs
• functional expression of GPCRs in eukaryotic hosts
structure determination of membrane
proteins
• membrane proteins are encoded by about 30% of all
genes
• 218 coordinate sets for membrane proteins deposited,
over 38,000 structure entries of soluble proteins in the
Protein Data Bank (Aug 2006)
• Hartmut Michel http://www.mpibpfrankfurt.mpg.de/michel/public/memprotstruct.html
• Martin Caffrey http://www.lipidat.chemistry.ohiostate.edu/MPDB/index.asp or http://www.mpdb.ul.ie\par
• Steve White
http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html
structure determination of membrane
proteins – 3D / X-ray
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Photosynthetic reaction centers, light-harvesting complexes
Cytochrome c oxidase
Cytochrome bc1 complexes, bc1 complex with cytochrome c, cytochrome b6f
Potassium channels (KcsA …), chloride channel from E. coli (ClC)
Mechanosensitive ion channels (MscL, MscS)
Bacteriorhodopsin, halorhodopsin, sensoryrhodopsin II, SRII-transducer
complex
Fumarate reductase / succinate dehydrogenase
F1Fo-ATPase (F1 and c subunits)
Sarcoplasmic reticulum calcium-ATPase
Rhodopsin
Aquaporin AQP1, glycerol facilitator GlpF
Photosystems I and II
Lipid A transporter from E. coli (MsbA), vitamin B12 uptake transporter from E.
coli (BtuCD)
Formate dehydrogenase-N from E. coli (Fdn-N)
AcrB multidrug exporter from E. coli
Lac permease (LacY), glycerol-3-phosphate transporter (GlpT)
Sec protein-conducting channel from Methanococcus jannaschii
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structure determination of membrane
proteins – 3D / X-ray
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FepA / FhuA / FecA / BtuB outer membrane iron transporters
BtuB bound with colicin
Porins (OmpF, PhoE, LamB, ScrY)
Alpha-hemolysin
Outer membrane phospholipase (OMPLA)
Outer membrane protease OmpT, OpcA
TolC channel
Outer membrane proteins OmpA, OmpX
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Prostaglandin H2 synthase
Squalene-hopene cyclase
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structure determination of membrane
proteins – 3D / X-ray
• mostly bacterial / archae-bacterial membrane protein
structures
• few eukaryotic membrane protein structures
– natural source (rhodopsin from retina, Ca-ATPase
from muscle)
– even fewer recombinant eukaryotic membrane
protein structures
structures of recombinant eukaryotic
membrane proteins
• rat voltage-gated potassium channel (methylotrophic
yeast Pichia pastoris) Long, MacKinnon 2005
• crystals of the rabbit calcium ATPase (S. cerevisiae)
Jidenko, Nissen 2005
• spinach aquaporin (Pichia pastoris) TörnrothHorsefield, Kjellbom 2006
• rat aquaporin (insect cell – baculovirus) Hiroaki,
Fujiyoshi 2006
integral membrane protein
▫ hydrophobic → detergents
▫ expression is often toxic for the host
▫ no universal expression system for all membrane proteins
insertion of membrane proteins into the
membrane
topology of integral membrane proteins
• Kim, von Heijne, PNAS 103: 11142, 2006, yeast
membrane proteome
• Cout ~ 20%
• Cin ~ 80%, even number of TMDs predominate
topology of integral membrane proteins
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• positive-inside rule (von Heijne, Nature 341: 456, 1989)
• hydrophobicity of TMD
• length of TMD
recognition of transmembrane helices by
the endoplasmic reticulum translocon
• Hessa, White, von Heijne:
Nature 433: 377-381, 2005
• in vitro assay to quantify the
efficiency with which
designed transmembrane
segments insert into dog
pancreas rough microsomes
recognition of transmembrane helices by
the endoplasmic reticulum translocon
derived from H-segments with the
indicated amino acid placed in the middle
of the 19-residue hydrophobic stretch
recognition of transmembrane helices by
the endoplasmic reticulum translocon
• ‘biological’ hydrophobicity scale, much in common
with hydrophobicity scales derived from biophysical
measurements
• implies that direct protein-lipid interactions are
involved in the recognition of TM helices by the
translocon
• strong dependence on sequence position of aromatic
and charged residues in TM segments
• can one ‘optimize’ membrane protein expression by
sequence analysis ?
structure of translocon
• v. d. Berg, Rapoport, Nature 427: 36, 2004
Methanococcus jannaschii
topics
• general considerations, mechanism of membrane
insertion, topology
• examples of overexpression of bacterial and
eukaryotic membrane proteins in various hosts
systems
• functional expression of G-protein-coupled receptors
(GPCRs) in E. coli
• factors influencing the expression levels and stability
of GPCRs
• functional expression of GPCRs in eukaryotic hosts
expression systems
• mammalian cells
• stable, transient transfection
• Semliki Forest Virus
• insect cells
• stable (Sf9, Drosophila Schneider S2)
• baculovirus system
• yeast
• chromosomal integration
• plasmids
• Escherichia coli, Lactococcus lactis
which expression system is best for highlevel production of functional receptors ?
• no universal expression system for all membrane
proteins
• trial and error approach
• but: membrane protein overexpression is possible !!
requirements for correct folding and
function of membrane protein
• post-translational modifications
• N-glycosylation
• disulphide bond formation
• lipid composition of host membrane
• molecular chaperones
• …
practical aspects
• maintenance of cell line
• may be difficult for stable mammalian cell lines
• easy for E. coli and yeast
• scale-up of expression
• yes: stable mammalian cells, insect cells /
baculovirus, yeast, E. coli
• biological safety aspects
• cell breakage at large scale
• problematic in case of yeast ?
problems with expression systems
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mammalian cells
• incorrect folding has been shown
insect cells
• membrane protein may not be N-glycosylated
• high-mannose type glycosylation
• membranes with low levels of cholesterol
• large proportion of membrane protein may be incorrectly folded
yeast
• proteolysis
• targeting of membrane protein to vacuole
• ergosterol (no cholesterol)
Escherichia coli
• proteolysis
• inclusion body formation (refolding necessary)
• no N-glycosylation
• lack of cholesterol
why is membrane protein overexpression
toxic to host cells ?
• function of membrane protein itself (e.g. ion channel)
• pGluR: potassium selective glutamate receptor from
Synechocystis (M1-P-M2, N-out) cannot be expressed in E.
coli
• but: KcsA potassium channel from Streptomyces lividans
(M1-P-M2, N-in) can be overexpressed in E. coli
– Schrempf, EMBO J. 14: 5170, 1995
– Heginbotham, Biochem. 36: 10335, 1997; synthetic
gene, Eco high codon usage, N-terminal His tag, Cterminal Strep tag, pASK75 (tet promoter)
– Cortes, Biochem. 36: 10343, 1997; N-terminal His tag,
pQE vector
why is membrane protein overexpression
toxic to host cells ?
• constitutive activity of membrane protein can promote
intracellular signaling
– GPCR β2-adrenergic receptor in stable CHO cells
at 200 pmol/mg (20 Mio R/cell), constitutive
expression (Lohse, Naunyn-Schmiedeberg’s Arch.
Pharmacol. 345, 444, 1992) → cell line died
– but: GPCR calcitonin receptor in stable MEL cells,
integration-independent, erythroid-specific
expression from β-globin promoter, differentiation /
induction with DMSO → 60 pmol/mg (2 Mio R/cell)
(Needham, PEP 6, 124, 1995)
why is membrane protein overexpression
toxic to host cells ?
• presence of large amounts of membrane protein
could disturb membrane
– but: overexpression of fumarate reductase in E.
coli leads to additional intracellular membrane
systems containing ordered arrays of Frd (Weiner,
J. Bacteriol. 158: 590, 1984)
why is membrane protein overexpression
toxic to host cells ?
• T7 RNA polymerase expression system / BL21(DE3)
(Miroux and Walker, JMB 260: 289, 1996)
– protein overexpression is limited or prevented by
cell death
– selection procedure to allow high-level expression
of target proteins (deposited as inclusion bodies)
– high cell density, no toxic effect
• transcription, translation, membrane insertion or
inclusion body formation cannot be considered
separately
• no general cloning strategy that guarantees
overexpression of a given prokaryotic membrane
protein in E. coli
• Gunn, Tate, Henderson: sugar-H+ symport protein
FucP, Mol. Microbiol. 12: 799-809, 1994
topics
• general considerations, mechanism of membrane
insertion, topology
• examples of overexpression of bacterial and
eukaryotic membrane proteins in various hosts
systems
• functional expression of G-protein-coupled receptors
(GPCRs) in E. coli
• factors influencing the expression levels and stability
of GPCRs
• functional expression of GPCRs in eukaryotic hosts
G-protein-coupled receptors
(Palczewski et al., 2000)
(Okada et al., 2002)
(Li et al., 2004)
rat neurotensin receptor (NTR, NTS-1)
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424 aa, 47 kDa
rat brain cDNA library
Tanaka et al., Neuron 4, 847, 1990
cDNA from S. Nakanishi
• neurotensin
Glp-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-IleLeu
à NTR modulates dopaminergic neurons
à NTR is involved in pancreatic cancer
expression of NTR in eukaryotic cells
• insect cells / baculovirus (transient, NTR-H5M)
• 188000 R/cell or 0.02 mg/L
• MEL cells (stable, NTR-H6F)
• 455000 R/cell or 0.04 mg/L
• proteolysis of C-terminus
• CHO, pCyt-TS (stable, NTR-H10F)
• 360000 R/cell or 0.03 mg/L
expression of seven-helix G-protein coupled
receptors in Escherichia coli
practical aspects
• maintenance of “cell line” is easy
• scale-up of expression is possible
• cell breakage at large scale not problematic
tools for assessing expression levels
• functional receptors → ligand binding analysis
• total receptor protein → Western blot (‘tag’)
expression as maltose-binding protein
fusion
influence of tag on expression
Tucker & Grisshammer, 1996
expression in E. coli of the neurotensin
receptor fusion protein
expression levels of NTR fusion protein
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1000 receptors/cell ([3H]NT)
3-5 nmol/L of culture (0.3 - 0.5 mg/L)
9 pmol/mg of total solubilized protein
24 pmol/mg of membrane protein
expression levels of NTR fusion protein
• monitor some parameters
during expression
• pH, OD600 vs. radio-ligand
binding assay
• source of media
general applicability of expression system
• Cannabinoid CB1 receptor: No (degraded)
• Cannabinoid CB2 receptor: 17-39 pmol/mg
(MBP-CB2-HF) (Calandra et al., 1997)
• Substance K receptor: 7 pmol/mg
(MBP-SKR-HMTX) (Grisshammer et al., 1994)
• Neurotensin receptor: 24 pmol/mg
(MBP-T43NTR-TrxA-H10) (Grisshammer & Tucker, 1997)
• Adenosine A2a receptor: 17-34 pmol/mg
(MBP-A2aTr316-H10) (Weiß & Grisshammer, 2002)
topics
• general considerations, mechanism of membrane
insertion, topology
• examples of overexpression of bacterial and
eukaryotic membrane proteins in various hosts
systems
• functional expression of G-protein-coupled receptors
(GPCRs) in E. coli
• factors influencing the expression levels and stability
of GPCRs
• functional expression of GPCRs in eukaryotic hosts
expression of GPCRs in P. pastoris, insect
cells, SFV
• Andre, Pattus, Michel, Reinhart, Protein Science 15:
1115, 2006: 20 GPCRs in Pichia pastoris
• Akermoun, Gearing, PEP 44: 65, 2005: 16 GPCRs in
3 insect cell lines
• Hassaine, Lundstrom PEP 45: 343, 2006: SFV 101GPCRs
• even closely related proteins behave differently
• Western blot analysis vs. ligand binding: Western blot
signals do not allow any correlation with amount of
correctly folded receptors
summary
Ö membrane protein overexpression is possible
Ö membrane proteins show individuality
Ö bacterial targets best made in E. coli
Ö eukaryotic targets best made in eukaryotic hosts
(exception: some GPCRs …)
Ö analysis mode for functionality