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Heterologous protein production in Escherichia coli : strategies and challenges

Heterologous protein production in Escherichia coli : strategies and challenges. François Baneyx Department of Chemical Engineering and Bioengineering University of Washington, Seattle WA. Bottlenecks to efficient protein expression in E. coli. l. Inefficient transcription.

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Heterologous protein production in Escherichia coli : strategies and challenges

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  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 l Inefficient transcription No or little protein synthesized u Promoter choice and design l Inefficient translation No or little protein synthesized u Codon usage Transcript stability Transcript secondary structure u u Aggregation or degradation l Inefficient folding (cytoplasmic or periplasmic) u Improper secondary, tertiary or quaternary structure formation Inefficient or improper disulfide bridge formation Inefficient isomerization of peptidyl-prolyl bonds u u Aggregation or degradation l Inefficient membrane insertion/translocation l Toxicity Cell death

  3. Intracellular environment and protein synthesis Escherichia coli

  4. Folding modulators of E. coli l Molecular chaperonesare a class of proteins that help other polypeptides fold or reach a proper cellular location without becoming part of the final structure Many - but not all - cytoplasmic chaperones are heat shock proteins transcribed at high level by Es32 The s32 regulon consists of ≈ 30 heat shock proteases and chaperones: u DnaK-DnaJ-GrpE (Hsp70/40 family) GroEL-GroES (Hsp60/10 family) ClpB (Hsp100 family) HtpG (Hsp90 family) IbpA-IbpB (sHsp family) Hsp33 Hsp31 “Folding” chaperones u u “Disaggregating” chaperone u ? u “Holding” chaperones u u l Foldasesare a class of proteins that accelerate rate-limiting steps along the folding pathway Thiol/disulfide oxidoreductases catalyze disulfide formation and isomerization Peptidyl-prolyl cis/trans isomerases catalyze the trans to cis isomerization of X-Pro bonds

  5. J K K T F J ATP 3' 5' GrpE ATP GrpE ADP ADP Native Aggregate ADP ATP GroEL GroES Folding chaperones in de novo folding

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

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

  8. However, this strategy does not always work

  9. a a a J K K T F J ATP 3' 5' GrpE ATP ADP ADP Disaggregation Native ADP IbpA/B ATP Holding Hsp31 GroEL GroES ClpB Hsp33 Chaperone-assisted protein folding GrpE

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

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

  12. The cold-shock response and CspA l Transfer of growing cells from 37 to 10-15oC triggers the cold shock response 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

  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. A combination of cspA-driven transcription and DnaK/J co-expression transiently increases IL21 solubility pMM101 + pTG10 pMM101 + pDnaK/J pMM101 + pGroESL

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

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

  19. 37ºC to A600 ≈ 0.4 1h at 37ºC 37ºC IPTG 15ºC IAA IAA IAA Effect of mutant thioredoxin co-expression on the recovery of active MalG17-PhoA in wt, trxB and trxB gor cells Wild type trxB trxB gor

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

  21. Acknowledgments l Dr. Jeff Thomas Paulene Quigley Dr. Jess Vasina Wim 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 l l l l l l l l l l l l l

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