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A High-Throughput Automated Protein Folding System

Part of the Methods in Molecular Biology book series (MIMB,volume 2025)

Abstract

In vitro protein folding can be employed to produce complex proteins expressed as insoluble inclusion bodies in E. coli from laboratory to commercial scale. Often the most challenging step is identification of renaturation conditions that will enable the denatured protein to form the native structure at an acceptable yield. Generally this requires screening a matrix of buffers and stabilizers to find an appropriate solution. Herein, we describe an automated and quantitative method to identify optimal in vitro protein folding parameters with a high rate of success.

Key words

  • High throughput
  • Automation
  • Protein folding
  • Screen
  • Liquid handling robot
  • Microfluidic capillary electrophoresis
  • E. coli

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  • DOI: 10.1007/978-1-4939-9624-7_6
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References

  1. Huang CJ, Lin H, Yang X (2012) Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements. J Ind Microbiol Biotechnol 39:383–399

    CAS  CrossRef  Google Scholar 

  2. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in microbial systems. Front Microbiol 5:341

    PubMed  PubMed Central  Google Scholar 

  3. Zhang J (2010) Mammalian cell culture for biopharmaceutical production. In: Baltz RH, Davies JE, Demain AL (eds) Manual of industrial microbiology and biotechnology, 3rd edn. ASM Press, Washington, DC, pp 157–178

    Google Scholar 

  4. Swietnicki W (2006) Folding aggregated proteins into functionally active forms. Curr Opin Biotechnol 17:367–372

    CAS  CrossRef  Google Scholar 

  5. Marston FAO, Lowe PA, Doel MT, Schoemaker JM, White S, Angal S (1984) Purification of calf prochymosin (prorennin) synthesized in Escherichia coli. Nat Biotechnol 2:800–804

    CAS  CrossRef  Google Scholar 

  6. Marston FAO (1986) The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem J 240:1–12

    CAS  CrossRef  Google Scholar 

  7. Mukhopadhyay A (1997) Inclusion bodies and purification of proteins in biologically active forms. Adv Biochem Eng Biotechnol 56:61–109

    CAS  PubMed  Google Scholar 

  8. Schoemaker JM, Brasnett AH, MFA O (1985) Examination of calf prochymosin accumulation in Escherichia coli: disulphide linkages are a structural component of prochymosin-containing inclusion bodies. EMBO J 4(3):775–780

    CAS  CrossRef  Google Scholar 

  9. Vallejo LF, Rinas U (2004) Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microb Cell Factories 3:11

    CrossRef  Google Scholar 

  10. Fischer B, Sumner I, Goodenough P (1993) Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion bodies. Biotechnol Bioeng 41(1):3–13

    CAS  CrossRef  Google Scholar 

  11. Rudolph R, Lilie H (1996) In vitro folding of inclusion body proteins. FASEB J 10(1):49–56

    CAS  CrossRef  Google Scholar 

  12. Clark EDB (1998) Refolding of recombinant proteins. Curr Opin Biotechnol 9:157–163

    CAS  CrossRef  Google Scholar 

  13. Middleberg APJ (2002) Preparative protein folding. Trends Biotechnol 10:437–443

    CrossRef  Google Scholar 

  14. Coutard B, Danchin EGJ, Oubelaid R, Canard B, Bignon C (2012) Single pH buffer refolding screen for protein from inclusion bodies. Protein Expr Purif 82(2):352–359

    CAS  CrossRef  Google Scholar 

  15. Dechavanne V, Barrillat N, Borlat F, Hermant A, Magnenat L, Paquet M, Antonsson B, Chevalet L (2011) A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions. Protein Expr Purif 75(2):192–203

    CAS  CrossRef  Google Scholar 

  16. Qoronfleh MW, Hesterberg LK, Seefeldt MB (2007) Confronting high-throughput protein refolding using high pressure and solution screens. Protein Expr Purif 55(2):209–224

    CAS  CrossRef  Google Scholar 

  17. Buswell AM, Ebtinger M, Vertes AA, Middleberg APJ (2002) Effect of operating variables on the yield of recombinant trypsinogen for a pulse-fed dilution-refolding reactor. Biotechnol Bioeng 77(4):435–444

    CAS  CrossRef  Google Scholar 

  18. Lin L, Seehra J, Stahl ML (2006) High-throughput identification of refolding conditions for LXRbeta without a functional assay. Protein Expr Purif 47(2):355–366

    CAS  CrossRef  Google Scholar 

  19. Vincentelli R, Canaan S, Campanacci V, Valencia C, Maurin D, Frassinetti F, Scappucini-Calvo L, Bourne Y, Cambillau C, Bignon C (2004) High-throughput automated refolding screening of inclusion bodies. Protein Sci 13(10):2782–2792

    CAS  CrossRef  Google Scholar 

  20. Scheich C, Niesen FH, Seckler R, Bussow K (2004) An automated in vitro protein folding screen applied to a human dynactin subunit. Protein Sci 13(2):370–380

    CAS  CrossRef  Google Scholar 

  21. Tobbell DA, Middleton BJ, Raines S, Needham MRC, Taylor IWF, Beveridge JY, Abbott WM (2002) Identification of in vitro folding conditions for procathepsin S and cathepsin S using fractional factorial screens. Protein Expr Purif 24(2):242–254

    CAS  CrossRef  Google Scholar 

  22. Armstrong N, De Lencastre A, Gouaux E (1999) A new protein folding screen: application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase. Protein Sci 8(7):1475–1483

    CAS  CrossRef  Google Scholar 

  23. Walther C, Mayer S, Jungbauer A, Dürauer A (2014) Getting ready for PAT: Scale up and inline monitoring of protein refolding of Npro fusion proteins. Process Biochem 49(7):1113–1121

    CAS  CrossRef  Google Scholar 

  24. Lee SH, Carpenter JF, Chang BS, Randolph TW, Kim YS (2006) Effects of solutes on solubilization and refolding of proteins from inclusion bodies with high hydrostatic pressure. Protein Sci 15(2):304–313

    CAS  CrossRef  Google Scholar 

  25. An P, Winters D, Walker KW (2016) Automated high-throughput dense matrix protein folding screen using a liquid handling robot combined with microfluidic capillary electrophoresis. Protein Expres Purif 120:138–147

    CAS  CrossRef  Google Scholar 

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Acknowledgments

We would like to acknowledge Alex Mladenovic and Randy Hecht for contributing to the construction and programming of the Biomek, Tom Boone for protein folding condition guidance and Jeff Lewis for expressing the recombinant protein described in this chapter.

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Correspondence to Kenneth W. Walker .

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Walker, K.W., An, P., Winters, D. (2019). A High-Throughput Automated Protein Folding System. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9624-7_6

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  • DOI: https://doi.org/10.1007/978-1-4939-9624-7_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9623-0

  • Online ISBN: 978-1-4939-9624-7

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