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Cited by 34Monoclonal and Polyclonal Antibodies ResearchProtein purification and stabilityBacterial Genetics and Biotechnology

Enhancement of antibody fragment secretion into the Escherichia coli periplasm by co-expression with the peptidyl prolyl isomerase, FkpA, in the cytoplasm

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Levy, Raphael, Ahluwalia, Kiran, Bohmann, David J., Giang, Hoa M., Schwimmer, Lauren J., Issafras, Hassan, Reddy, Nithin B., Chan, Chung +2 more·2013·Journal of Immunological Methods
DOI 10.1016/j.jim.2013.04.010
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Abstract

Improper protein folding or aggregation can frequently be responsible for low expression and poor functional activity of antibody fragments secreted into the Escherichia coli periplasm. Expression issues also can affect selection of antibody candidates from phage libraries, since antibody fragments displayed on phage also are secreted into the E. coli periplasm. To improve secretion of properly folded antibody fragments into the periplasm, we have developed a novel approach that involves co-expressing the antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, lacking its signal sequence (cytFkpA) which consequently is expressed in the E. coli cytosol. Cytoplasmic expression of cytFkpA improved secretion of functional Fab fragments into the periplasm, exceeding even the benefits from co-expressing Fab fragments with native, FkpA localized in the periplasm. In addition, panning and subsequent screening of large Fab and scFv naïve phage libraries in the presence of cytFkpA significantly increased the number of unique clones selected, as well as their functional expression levels and diversity.

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  1. Arie, J.P., Sassoon, N., et al., 2001. Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli. Mol. Microbiol. 39 (1), 199.
  2. Bach, H., Mazor, Y., et al., 2001. Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single- chain antibodies. J. Mol. Biol. 312 (1), 79.
  3. Baneyx, F., Mujacic, M., 2004. Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotechnol. 22 (11), 1399.
  4. Betton, J.M., Sassoon, N., et al., 1998. Degradation versus aggregation of misfolded maltose-binding protein in the periplasm of Escherichia coli. J. Biol. Chem. 273 (15), 8897.
  5. Bothmann, H., Pluckthun, A., 1998. Selection for a periplasmic factor improving phage display and functional periplasmic expression. Nat. Biotechnol. 16 (4), 376.
  6. Bothmann, H., Pluckthun, A., 2000. The periplasmic Escherichia coli peptidylprolyl cis, trans-isomerase FkpA. I. Increased functional expression of antibody fragments with and without cis-prolines. J. Biol. Chem. 275 (22), 17100.
  7. Dalal, K., Duong, F., 2011. The SecY complex: conducting the orchestra of protein translocation. Trends Cell Biol. 21 (9), 506.
  8. Duenas, M., Vazquez, J., et al., 1994. Intra-and extracellular expression of an scFv antibody fragment in E. coli: effect of bacterial strains and pathway engineering using GroES/L chaperonins. Biotechniques 16 (3), 476 (480-473).
  9. Feige, M.J., Hendershot, L.M., et al., 2010. How antibodies fold. Trends Biochem. Sci. 35 (4), 189.
  10. Forsberg, G., Forsgren, M., et al., 1997. Identification of framework residues in a secreted recombinant antibody fragment that control production level and localization in Escherichia coli. J. Biol. Chem. 272 (19), 12430.
  11. Georgiou, G., Telford, J.N., et al., 1986. Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase. Appl. Environ. Microbiol. 52 (5), 1157.
  12. Hayhurst, A., Harris, W.J., 1999. Escherichia coli skp chaperone coexpression improves solubility and phage display of single-chain antibody frag- ments. Protein Expr. Purif. 15 (3), 336.
  13. Heo, M.A., Kim, S.H., et al., 2006. Functional expression of single-chain variable fragment antibody against c-Met in the cytoplasm of Escherichia coli. Protein Expr. Purif. 47 (1), 203.
  14. Hesterkamp, T., Bukau, B., 1996. The Escherichia coli trigger factor. FEBS Lett. 389 (1), 32.
  15. Horne, S.M., Young, K.D., 1995. Escherichia coli and other species of the Enterobacteriaceae encode a protein similar to the family of Mip-like FK506-binding proteins. Arch. Microbiol. 163 (5), 357.
  16. Hu, X., O'Hara, L., et al., 2007. Optimisation of production of a domoic acid- binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support. Protein Expr. Purif. 52 (1), 194.
  17. Jurado, P., de Lorenzo, V., et al., 2006. Thioredoxin fusions increase folding of single chain Fv antibodies in the cytoplasm of Escherichia coli: evidence that chaperone activity is the prime effect of thioredoxin. J. Mol. Biol. 357 (1), 49.
  18. Knappik, A., Pluckthun, A., 1995. Engineered turns of a recombinant antibody improve its in vivo folding. Protein Eng. 8 (1), 81.
  19. Lee, H.C., Bernstein, H.D., 2002. Trigger factor retards protein export in Escherichia coli. J. Biol. Chem. 277 (45), 43527.
  20. Levy, R., Weiss, R., et al., 2001. Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones. Protein Expr. Purif. 23 (2), 338.
  21. Mavrangelos, C., Thiel, M., et al., 2001. Increased yield and activity of soluble single-chain antibody fragments by combining high-level expression and the Skp periplasmic chaperonin. Protein Expr. Purif. 23 (2), 289.
  22. Maynard, J., Adams, E.J., et al., 2005. High-level bacterial secretion of single- chain alphabeta T-cell receptors. J. Immunol. Methods 306 (1-2), 51.
  23. Missiakas, D., Betton, J.M., et al., 1996. New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/ OmpH. Mol. Microbiol. 21 (4), 871.
  24. Natale, P., Bruser, T., et al., 2008. Sec-and Tat-mediated protein secretion across the bacterial cytoplasmic membrane-distinct translocases and mechanisms. Biochim. Biophys. Acta 1778 (9), 1735.
  25. Padiolleau-Lefevre, S., Debat, H., et al., 2006. Expression of a functional scFv fragment of an anti-idiotypic antibody with a beta-lactam hydrolytic activity. Immunol. Lett. 103 (1), 39.
  26. Perez-Perez, J., Gutierrez, J., 1995. An arabinose-inducible expression vector, pAR3, compatible with ColE1-derived plasmids. Gene 158 (1), 141.
  27. Proba, K., Worn, A., et al., 1998. Antibody scFv fragments without disulfide bonds made by molecular evolution. J. Mol. Biol. 275 (2), 245.
  28. Ramm, K., Pluckthun, A., 2000. The periplasmic Escherichia coli peptidylprolyl cis, trans-isomerase FkpA. II. Isomerase-independent chaperone activity in vitro. J. Biol. Chem. 275 (22), 17106.
  29. Saul, F.A., Arie, J.P., et al., 2004. Structural and functional studies of FkpA from Escherichia coli, a cis/trans peptidyl-prolyl isomerase with chaperone activity. J. Mol. Biol. 335 (2), 595.
  30. Schwimmer, L.J., Huang, B., et al., 2013. Discovery of diverse and functional antibodies from large human repertoire antibody libraries. J. Immunol. Methods 391 (1-2), 60.
  31. Soltes, G., Barker, H., et al., 2003. A new helper phage and phagemid vector system improves viral display of antibody Fab fragments and avoids propagation of insert-less virions. J. Immunol. Methods 274 (1-2), 233.
  32. Sonoda, H., Kumada, Y., et al., 2010. Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones. Protein Expr. Purif. 70 (2), 248.
  33. Soos, M.A., Siddle, K., et al., 1986. Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor. Biochem. J. 235 (1), 199.
  34. Strachan, G., Williams, S., et al., 1999. Reduced toxicity of expression, in Escherichia coli, of antipollutant antibody fragments and their use as sensitive diagnostic molecules. J. Appl. Microbiol. 87 (3), 410.
  35. Suominen, I., Karp, M., et al., 1987. Extracellular production of cloned alpha- amylase by Escherichia coli. Gene 61 (2), 165.
  36. Volokhina, E.B., Grijpstra, J., et al., 2011. Role of the periplasmic chaperones Skp, SurA, and DegQ in outer membrane protein biogenesis in Neisseria meningitidis. J. Bacteriol. 193 (7), 1612.
  37. Wall, J.G., Pluckthun, A., 1995. Effects of overexpressing folding modulators on the in vivo folding of heterologous proteins in Escherichia coli. Curr. Opin. Biotechnol. 6 (5), 507.
  38. Walton, T.A., Sandoval, C.M., et al., 2009. The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains. Proc. Natl. Acad. Sci. U. S. A. 106 (6), 1772.
  39. Worn, A., Pluckthun, A., 1998. Mutual stabilization of VL and VH in single- chain antibody fragments, investigated with mutants engineered for stability. Biochemistry 37 (38), 13120.
  40. Zhang, Z., Song, L.P., et al., 2003. Production of soluble and functional engineered antibodies in Escherichia coli improved by FkpA. Biotechniques 35 (5), 1032 (1041-1032).

Cited by

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Authors
Levy, R., Ahluwalia, K., Bohmann, D. J., Giang, H. M., Schwimmer, L. J., Issafras, H., Reddy, N. B., Chan, C., Horwitz, A. H., & Takeuchi, T.
Affiliations
XOMA (United States) (US)
Corresponding author
Levy, Raphael
ORCID
Venue
Journal of Immunological Methods
Publisher
Elsevier BV
Published
2013-08
Volume
394
Issue
1-2
Pages
10-21
ISSN
0022-1759 (ISSN-L)
DOI
10.1016/j.jim.2013.04.010
OpenAlex
W2169633028
Cited by
34
References
1
Citations by year
2026: 1 · 2025: 1 · 2024: 1 · 2023: 2 · 2022: 3 · 2021: 5 · 2020: 6 · 2019: 4 · 2018: 3 · 2016: 2 · 2015: 1 · 2014: 3 · 2013: 2
FWCI
2.07
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Immunological Methods (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
periplasmic secretion, FkpA, peptidyl-prolyl isomerase, Fab fragment, scFv, co-expression, protein folding, protein aggregation, phage display, antibody selection, Escherichia coli
MeSH terms
Enzyme-Linked Immunosorbent Assay, Escherichia coli, Immunoglobulin Fab Fragments, Membrane Proteins, Protein Folding, Peptide Library, Peptidylprolyl Isomerase, Periplasm, Escherichia coli Proteins

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