Acting on Folding Effectors to Improve Recombinant Protein Yields and Functional Quality

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1586)

Abstract

Molecular and chemical chaperones/foldases can strongly contribute to improve the amounts and the structural quality of recombinant proteins. Several methodologies have been proposed to optimize their beneficial effects. This chapter presents a condensed summary of the biotechnological opportunities offered by this approach followed by a protocol describing the method we use for expressing disulfide bond-dependent recombinant antibodies in the cytoplasm of bacteria engineered to overexpress sulfhydryl oxidase and DsbC isomerase. The system is based on the possibility to trigger the foldase expression independently and before the induction of the target protein. As a consequence, the recombinant antibody synthesis starts only after enough foldases have accumulated to promote correct folding of the antibody.

Key words

Molecular chaperones Disulfide isomerases Sulfhydryl oxidase Osmolytes Protein soluble aggregates Protein quality assessment Secretion efficiency 

References

  1. 1.
    Anfinsen CB (1972) The formation and stabilization of protein structure. Biochem J 128:737–749CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Taipale M, Krykbaeva I, Koeva M et al (2012) Quantitative analysis of HSP90-client interactions reveals principles of substrate recognition. Cell 150:987–1001CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Kerner MJ, Naylor DJ, Ishihama Y et al (2005) Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 122:209–220CrossRefPubMedGoogle Scholar
  4. 4.
    de Marco A, Vigh L, Diamant S et al (2005) Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones. Cell Stress Chaperones 10:329–339CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    de Marco A, Deuerling E, Mogk A et al (2007) Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli. BMC Biotechnol 7:32CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    de Marco A (2007) Protocol for preparing proteins with improved solubility by co-expression with molecular chaperones in Escherichia coli. Nat Protoc 2:2632–2639CrossRefPubMedGoogle Scholar
  7. 7.
    Diamant S, Rosenthal D, Azem A (2003) Dicarboxylic amino acids and glycine-betaine regulate chaperone-mediated protein disaggregation under stress. Mol Microbiol 49:401–410CrossRefPubMedGoogle Scholar
  8. 8.
    Schultz T, Liu J, Capasso P et al (2007) The solubility of recombinant proteins expressed in Escherichia coli is increased by otsA and otsB co-transformation. Biochem Biophys Res Commun 355:234–239CrossRefPubMedGoogle Scholar
  9. 9.
    Bandyopadhyay A, Saxena K, Kasturia N et al (2012) Chemical chaperones assist intracellular folding to buffer mutational variations. Nat Chem Biol 8:238–245CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Diamant S, Eliahu N, Rosenthal D et al (2001) Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses. J Biol Chem 276:39586–39591CrossRefPubMedGoogle Scholar
  11. 11.
    de Marco A (2014) Osmolytes as chemical chaperones to use in protein biotechnology. In: Doglia SM, Lotti M (eds) Protein aggregation in bacteria: functional and structural properties of inclusion bodies in bacterial cells. Wiley, Hoboken, NJ, pp 77–92CrossRefGoogle Scholar
  12. 12.
    Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17:353–358CrossRefPubMedGoogle Scholar
  13. 13.
    Swalley SE, Fulghum JR, Chambers SP (2006) Screening factors effecting a response in soluble protein expression: formalized approach using design of experiments. Anal Biochem 351:122–127CrossRefPubMedGoogle Scholar
  14. 14.
    Bora N, Bawa Z, Bill RM et al (2012) The implementation of a design of experiments strategy to increase recombinant protein yields in yeast (review). Methods Mol Biol 866:115–127CrossRefPubMedGoogle Scholar
  15. 15.
    Nozach H, Fruchart-Gaillard C, Fenaille F (2013) High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli. Microb Cell Fact 12:37CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Djender S, Schneider A, Beugnet A et al (2014) Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies. Microb Cell Fact 13:140CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Veggiani G, de Marco A (2011) Improved quantitative and qualitative production of single-domain intrabodies mediated by the co-expression of Erv1p sulfhydryl oxidase. Protein Expr Purif 79:111–114CrossRefPubMedGoogle Scholar
  18. 18.
    Raynal B, Lenormand P, Baron B et al (2014) Quality assessment and optimization of purified protein samples: why and how? Microb Cell Fact 13:180CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Nguyen VD, Hatahet F, Salo KE et al (2011) Pre-expression of a sulfhydryl-oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E. coli. Microb Cell Fact 10:1CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Liu JL, Zabetakis D, Walper SA et al (2014) Bioconjugates of rhizavidin with single domain antibodies as bifunctional immunoreagents. J Immunol Methods 411:37–42CrossRefPubMedGoogle Scholar
  21. 21.
    de Marco A (2015) Recombinant antibody production evolves into multiple options aimed at yielding reagents suitable for application-specific needs. Microb Cell Fact 14:125Google Scholar
  22. 22.
    Sala E, de Marco A (2010) Screening optimized protein purification protocols by coupling small-scale expression and mini-size exclusion chromatography. Protein Expr Purif 74:231–235CrossRefPubMedGoogle Scholar
  23. 23.
    Nominé Y, Ristriani T, Laurent C et al (2001) A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoprotein. Protein Eng 14:297–305CrossRefPubMedGoogle Scholar
  24. 24.
    Capasso P, Aliprandi M, Ossolengo G et al (2009) Monodispersity of recombinant Cre recombinase correlates with its effectiveness in vivo. BMC Biotechnol 9:80CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Biomedical Sciences and EngineeringUniversity of Nova GoricaVipavaSlovenia

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