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1. Simon Davis, Ed Evans: 21336 www.t-cellbiology.org/teaching.htm

2. Methods Course, October 25th Expression cloning In bacteria In mammalian cells Yeast 2-hybrid screens

3. Methods Course, October 25th Protein expression Why express proteins at all? How to decide on an expression strategy The methods - bacterial expression: the pET system - mammalian expression (transient, stable) 4. Judging protein quality The BIAcore (Ed Evans)

4. Expression cloning: lambda libraries Principle Construct cDNA library in bacteriophage expression vector, e.g. “l zap” or lgt11 Transduce (infect) bacterial host Plate out, allow plaques to form Transfer expressed protein to membrane Screen proteins with antibody (Western) Isolate clone from library

5. Expression cloning: lambda libraries plaque cDNA library plaque screen

6. Expression cloning: lambda libraries Advantage Not restricted to monomers or homodimers Disadvantages Most proteins do not fold spontaneously in bacteria, so need non-conformation sensitive antibodies (e.g. polyclonal antisera) So generally can’t use ligands to screen Will not automatically select full-length clones

7. Expression cloning with CDM8 Principle Transfect COS-1 cells with cDNA library in CDM8 Pan on antibody coated dish Isolate plasmid from bound cells, re-transfect 4. Repeat cycle 3-4 times 5. Transfect individual mini-preps to identify gene, then sequence

8. Expression cloning with CDM8

9. Expression cloning with CDM8 transiently expressed proteins antibody Cos-1 cell Y Y Y Y Y Y Y Y Y Y Y Y Y Y sequence clone

10. Expression cloning with CDM8 Advantages Can use monoclonal antibodies Isolates full length cDNAs Can potentially use ligands Disadvantages Only useful for monomers, homodimers etc

11. Expression cloning: 2-hybrid systems

12. Expression cloning: 2-hybrid systems Advantage Designed for identifying ligand-receptor pairs Disadvantages Only useful for protein pairs that fold well in the cytoplasm Likely to require a high affinity interaction of proteins X and Y Said to be difficult

13. Why express and study proteins? Proteins are of fundamental interest: biological systems are all about protein recognition An understanding of immunological phenomena increasingly depends on understanding how proteins behave

14. Why express and study proteins? Can expect hard answers to scientific questions: is this how my protein looks? Modern immunology is reagent-driven so the choice of protein can set the research agenda This can provide many opportunities for collaboration (i.e. lots of papers)

15. The expression strategy Cytosolic? Your protein • Bacterial expression • (e.g. pET vectors) • fast • often very large • amounts

16. The expression strategy Secreted or membrane bound? Your protein • 1. Bacterial re-folds • yields can be low (~1%) • refold conditions generally • differ for each protein • sparse-matrix screens are • available to help

17. The expression strategy Secreted or membrane bound? Your protein 2. Bacterial secretion systems - e.g. pET-12a,b,c vectors - yields often very low

18. The expression strategy Secreted or membrane bound? Your protein • Yeast (e.g. Pichia) • fast • very high yields • metabolic labelling (NMR) • deglycosylation possible • poor folding of • e.g. IgSF proteins needs to be glycosylated or don’t want to refold?

19. The expression strategy Secreted or membrane bound? Your protein needs to be glycosylated or don’t want to refold? 2. Baculovirus • can be very slow • modest yields: 1-5 mg/l • very good for some proteins e.g. MHC II

20. The expression strategy Secreted or membrane bound? Your protein 3. Mammalian cells (e.g. CHO K1 cells) • moderately fast • very high yields potentially (<400 mg/l) • sugars can be removed • (Lec3.2.8.1 cells) • transient or stable needs to be glycosylated or don’t want to refold?

21. Bacterial expression Basic features • proteins are expressed in the cytoplasm or secreted into the periplasmic space • periplasmic expression levels are very low • very few cell surface or secreted proteins fold in the cytosol • so most proteins of interest form inclusion bodies and have to be refolded

22. Bacterial expression Recommended: pET vectors - T7 promoter-based systems

23. Mammalian expression Basic features • expressed proteins are generally secreted, but can be put on the cell surface or made intracellularly • soluble expression is achieved by inserting stop codon immediately before the TM domain • proteins are glycosylated; refolding unnecessary • the more “intact” the protein, the better • fusion proteins, his-tagged proteins can be made

24. Transient expression • Advantages • transient expression takes 3-5 days • excellent for testing constructs • various fusion partners • transfection with CaPO4 or lipids (fast) • Disadvantage • beware of Fc fusion proteins - Fc folds very efficiently, possibly taking mis-folded protein with it

25. Transient expression Recommended: - EF1a-based expression vectors

26. Stable expression • The GS system • CHO cells transfected with CaPO4 or lipids • selection is via the glutamine synthetase (GS) gene • CHO cells have their own GS gene and can be killed with GS inhibitor, methionine sulphoximine • cells with extra GS from the plasmid survive higher levels of MSX than the mocks • expression is driven by strong hCMV promoter

27. Stable expression • The GS system, cont. • selection takes 2 weeks • potentially prodigious expression levels • Can make enough protein to thoroughly confirm that it’s OK • mutant CHO cells can be used to alter glycosylation, e.g. Lec3.2.8.1 cells • NO DISADVANTAGES

28. The glutamine synthetase-based gene expression system glutamine synthetase gene 5 ml clone 4A tcs 5 ml control tcs 2 mg CD4 AmpR pEE14.hcmv-GS 10.4 kb SV40 promoter hCMV promoter poly A 400 mgs/litre = 10 x “Harry Ward” units expressed protein

29. Expression of rat sCD2 for structural studies in CHO mutant Lec3.2.8.1 cells Deglycosylated sCD2 crystals Lec3.2.8.1 + endo H Lec3.2.8.1 CHO-K1

30. Is my protein any good? • Good signs • it’s expressed at high levels • if cys-containing, it runs at the right size on non-reducing SDS-PAGE • the protein is stable/active for days/weeks at 4ºC • the protein binds mAbs stoichiometrically (Westerns and ELISAs are not suitable for this) • the protein is soluble at high concentrations

31. Is my protein any good? • Good signs, cont. • the protein is non-aggregated according to gel filtration - the absolute key for doing BIAcore experiments and structural studies properly