1. Heterologous protein production in Escherichia coli: strategies and challenges François Baneyx Department of Chemical Engineering and Bioengineering University of Washington, Seattle WA

2. Bottlenecks to efficient protein expression in E. coli Promoter choice and design Inefficient transcriptionNo or little protein synthesized Codon usage Transcript stability Transcript secondary structure Improper secondary, tertiary or quaternary structure formation Inefficient or improper disulfide bridge formation Inefficient isomerization of peptidyl-prolyl bonds Inefficient translationNo or little protein synthesized Inefficient folding (cytoplasmic or periplasmic) Inefficient membrane insertion/translocation ToxicityCell death l l l l l u u u u u u u Aggregation or degradation

3. Escherichia coli Intracellular environment and protein synthesis

4. Folding modulators of E. coli Molecular chaperones are a class of proteins that help other polypeptides fold or reach a proper cellular location without becoming part of the final structure l Many - but not all - cytoplasmic chaperones are heat shock proteins transcribed at high level by E  32 The  32 regulon consists of ≈ 30 heat shock proteases and chaperones: DnaK-DnaJ-GrpE (Hsp70/40 family) GroEL-GroES (Hsp60/10 family) ClpB (Hsp100 family) HtpG (Hsp90 family) IbpA-IbpB (sHsp family) Hsp33 Hsp31 Foldases are a class of proteins that accelerate rate-limiting steps along the folding pathway l Thiol/disulfide oxidoreductases catalyze disulfide formation and isomerization Peptidyl-prolyl cis/trans isomerases catalyze the trans to cis isomerization of X-Pro bonds u u u u u u u “Folding” chaperones “Disaggregating” chaperone “Holding” chaperones ?

5. Folding chaperones in de novo folding Aggregate 3' 5' K TF J Native K ADP GrpE J GroEL GroES ATP ADP ATP ADP GrpE

6. DnaK-DnaJ co-expression and low temperatures improve preS2-S’-  -galactosidase folding 30ºC 37ºC 42ºC tacpreS2lacZS’

7. GroEL-GroES co-expression and low temperatures improve leptin folding

8. However, this strategy does not always work

9. aa a 3' 5' K TF J Native K ADP GrpE J GroEL GroES ATP ADP ATP ADP GrpE IbpA/B Hsp33 Hsp31 ClpB Disaggregation Holding Chaperone-assisted protein folding

10. E. coli Hsp31 mechanism of action T decrease T increase

11. To His-tag or not to His-tag...

12. Transfer of growing cells from 37 to 10-15 o C triggers the cold shock response l Cells growth and protein synthesis stop and resume at lower rates after 1-4h A subset of cold shock proteins (CspA, CspB, CspG, CspI, CsdA, RbfA) is induced over 10-fold A subset of proteins involved in housekeeping transcriptional/translational control (IF-2, NusA, HN-S, Pnp, GyrA, RecA) is induced 2-10-fold The cold-shock response and CspA

13. Use of rbfA mutants abolishes cspA promoter repression

14. cspA-driven transcription allows the production of a toxic and proteolytically-sensitive protein in full-length form

15. cspA-driven transcription allows the production of a poorly translated protein in a partially soluble form

16. pMM101 + pTG10pMM101 + pDnaK/JpMM101 + pGroESL A combination of cspA-driven transcription and DnaK/J co-expression transiently increases IL21 solubility

17. Making disulfide bridges in the E. coli cytoplasm Stable disulfide bonds form in the cytoplasm of surprisingly healthy trxB and trxB gor (sup) strains Incubation of trxB cells at low temperatures greatly increases oxidation efficiency Certain active site mutants of thioredoxin 1 (trxA) are able complement a null mutation in yeast PDI Purified thioredoxin exhibits PDI activity in vitro

18. ColE1-compatible plasmids for cspA-driven synthesis of thioredoxin 1 active site mutants

19. 37ºC to A 600 ≈ 0.4 IPTG 1h at 37ºC 37ºC 15ºC IAA Wild typetrxBtrxB gor Effect of mutant thioredoxin co-expression on the recovery of active MalG17-PhoA in wt, trxB and trxB gor cells

20. Low temperature co-expression of thioredoxin 1 CGHC mutant in an oxidizing background enhances IL21 solubility and stability

21. Acknowledgments Dr. Jeff Thomas Paulene Quigley Dr. Jess VasinaWim Hol Mirna Mujacic Dr. Kerri Cooper Joanne Palumbo Stephanie Richardson Dr. Konstantin Korotkov Yan Brodsky Dr. M.S.R. Sastry National Science Foundation American Cancer Society ZymoGenetics