Expression of membrane proteins for structure determination

Transcription

1 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

2 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

3 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

4 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

5 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 Martin Caffrey or Steve White

6 structure determination of membrane proteins 3D / X-ray 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

7 structure determination of membrane proteins 3D / X-ray 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 Prostaglandin H2 synthase Squalene-hopene cyclase

8 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

9 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) Törnroth- Horsefield, Kjellbom 2006 rat aquaporin (insect cell baculovirus) Hiroaki, Fujiyoshi 2006

10 integral membrane protein hydrophobic detergents expression is often toxic for the host no universal expression system for all membrane proteins

11 insertion of membrane proteins into the membrane

12 topology of integral membrane proteins Kim, von Heijne, PNAS 103: 11142, 2006, yeast membrane proteome C out ~ 20% C in ~ 80%, even number of TMDs predominate

13 topology of integral membrane proteins positive-inside rule (von Heijne, Nature 341: 456, 1989) hydrophobicity of TMD length of TMD

14 recognition of transmembrane helices by the endoplasmic reticulum translocon Hessa, White, von Heijne: Nature 433: , 2005 in vitro assay to quantify the efficiency with which designed transmembrane segments insert into dog pancreas rough microsomes

15 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

16 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?

17 structure of translocon v. d. Berg, Rapoport, Nature 427: 36, 2004 Methanococcus jannaschii

18 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

19 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

20 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!!

21 requirements for correct folding and function of membrane protein post-translational modifications N-glycosylation disulphide bond formation lipid composition of host membrane molecular chaperones

22 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?

23 problems with expression systems 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

24 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, C- terminal Strep tag, pask75 (tet promoter) Cortes, Biochem. 36: 10343, 1997; N-terminal His tag, pqe vector

25 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)

26 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)

27 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

28 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: , 1994

29 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

30 G-protein-coupled receptors (Palczewski et al., 2000) (Okada et al., 2002) (Li et al., 2004)

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32 rat neurotensin receptor (NTR, NTS-1) 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-Ile- Leu NTR modulates dopaminergic neurons NTR is involved in pancreatic cancer

33 expression of NTR in eukaryotic cells insect cells / baculovirus (transient, NTR-H5M) R/cell or 0.02 mg/l MEL cells (stable, NTR-H6F) R/cell or 0.04 mg/l proteolysis of C-terminus CHO, pcyt-ts (stable, NTR-H10F) R/cell or 0.03 mg/l

34 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

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36 tools for assessing expression levels functional receptors ligand binding analysis total receptor protein Western blot ( tag )

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38 expression as maltose-binding protein fusion

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40 influence of tag on expression Tucker & Grisshammer, 1996

41 expression in E. coli of the neurotensin receptor fusion protein

42 expression levels of NTR fusion protein 1000 receptors/cell ([ 3 H]NT) 3-5 nmol/l of culture ( mg/l) 9 pmol/mg of total solubilized protein 24 pmol/mg of membrane protein

43 expression levels of NTR fusion protein monitor some parameters during expression ph, OD 600 vs. radio-ligand binding assay source of media

44 general applicability of expression system Cannabinoid CB1 receptor: No (degraded) Cannabinoid CB2 receptor: 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: pmol/mg (MBP-A2aTr316-H10) (Weiß & Grisshammer, 2002)

45 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

46 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 101- GPCRs 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

47 summary membrane protein overexpression is possible membrane proteins show individuality bacterial targets best made in E. coli eukaryotic targets best made in eukaryotic hosts (exception: some GPCRs ) analysis mode for functionality