1. Expressing disulphide-rich (recombinant) proteins in E. coli Kovilen Sawmynaden Email: ks01@imperial.ac.uk

2. 2 Native disulphide bond formation Disulphide bonds are integral to the structure and function of extracellular proteins Requires cysteine oxidation In E. coli, native disulphide bond formation takes place within the periplasmic space Disulphides are formed in vivo in disulphide exchange reactions e.g. Dsb family The cytoplasm has a more negative redox potential and maintained ‘reducing’ by the thioredoxin (trxB) and glutaredoxin pathways (gor) (Stewart et. al., 1998 )

3. 3 A recombinant problem: taking the MIC! What is the recombinant problem? MIC proteins from T. gondii form surface adhesins containing multiple cysteine residues For example, MIC6 contains 20 cysteines, 18 of which form 9 disulphides… Expression tests of numerous MIC proteins showed that little or no protein was obtainable using standard protocols Indicative that disulphides were not forming correctly MRLFR CC AAAVVAAESLLWLKNGSPFFAFLPGNGEIAD N C SGNP C GGTAAGT C INTPSGYD C R C EPGYVLGVEN DQVT C MMPSGVPMANFVQLSETPAA C SSNP C GPEAA GT C KETNSGYI C R C NQGYRISLDGTGNVT C IVRQESG C EENG C GPPDAVQS C RRLTGTAGRL C V C KENFIATIDAS AHIT C KRVPPHYRKPPFEFGKGGHPVDSEPSKRQREDE GESREPESDSTEPGRDQERRTPLEESQEPEGSTPDSQQ SRGGSGSDSTESEEQGKEREEGSGHAGAIAGGVIGGLLL LSAAGAGVAYMRKSGSGGGEEIEYERGIEAAEASEVEVL VDLDSKTWD MIC6 disulphide-rich primary sequence

4. 4 Origami TM and a role reversal for thioredoxin Can we overcome reductive properties of cytoplasm? Commercially available strains that have mutations in trx B and gor e.g. Origami™ (DE3) (Novagen) Selectable on kanamycin and tetracycline 10-fold increase in functional protein in alkaline phosphatase study (Prinz et al. 1997) Thioredoxin can catalyse disulphide bond formation, in a reducing background (Stewart et al., 1998 ) Commercially available pET-32 vector contains a cleavable thioredoxin (trx A) tag (Novagen)

5. 5 Expression and purification protocol Induce protein expression with IPTG for 6hrs at 30° Standard IMAC (using His-tag) Cleave thioredoxin tag using factor Xa at room temperature for 6-8hrs Inactivation (1mmM AEBSF) and removal (Xarrest™ agarose) of factor Xa Additional IMAC to remove thioredoxin tag Milligram quantities (typically 0.1M-1mM final concentration) suitable for structural studies Marker + wash fractions… …eluted fusion protein IMAC Marker + hourly samples… …fusion protein …thioredoxin …target protein Factor Xa cleavage reaction

6. 6 Evidence for disulphide bridges MIC1 M2AP +DTT- DTT+DTT - DTT 1.SDS-PAGE 4.Functional Blumenschein et al. (2007) 3.Structure 2.Folded

7. 7 Further cloning and expression options 1.Factor Xa, Enterokinase and thrombin cleavage options 2.Origami TM or Rosetta-gami TM expression strains – carries a chloramphenicol resistant plasmid encoding 7 rare tRNAs in addition to trx and gor mutations

8. 8 Co-express two proteins containing disulphide bonds LIC Duet™ Adaptors (Novagen) allow co-expression of two ORFs into one bacterial expression vector Provides additional T7 promoter sequence and thioredoxin tag (but no His-tag) IMAC as before, two over- expressed proteins if complex formed Advantages include in vivo folding and/or binding event and potential for increased yield MIC1 MIC6

9. 9 Summary Challenges of expressing disulphide-rich proteins arise from reducing conditions of the cytoplasm This can be overcome by using engineered strains carrying mutations in cytoplasmic reductases (trxB and gor) System is especially powerful when combined with a thioredoxin fusion partner Can be extended to a co-expression system for 2 (or more) disulphide- rich proteins We have used this overall approach to successfully express proteins containing up to 16 cysteines Saouros et al. (2007) Protein and Peptide Letters In press Acknowledgement and thanks to all members of the group associated with the MIC project in Steve Matthews’ lab!