Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Improving the Productivity of Single-Chain Fv Antibody Against c-Met by Rearranging the Order of its Variable Domains

  • Kim, Yu-Jin (Department of Polymer Science and Chemical Engineering, Pusan National University) ;
  • Neelamegam, Rameshkumar (Department of Polymer Science and Chemical Engineering, Pusan National University) ;
  • Heo, Mi-Ae (Department of Polymer Science and Chemical Engineering, Pusan National University) ;
  • Edwardraja, Selvakumar (Department of Polymer Science and Chemical Engineering, Pusan National University) ;
  • Paik, Hyun-Jong (Department of Polymer Science and Chemical Engineering, Pusan National University) ;
  • Lee, Sun-Gu (Department of Polymer Science and Chemical Engineering, Pusan National University)
  • Published : 2008.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

Single-chain Fv (scFv) antibody against c-Met is expected to be employed in clinical treatment or imaging of cancer cells owing to the important biological roles of c-Met in the proliferation of malignancies. Here, we show that the productivity of scFv against c-Met in Escherichia coli is significantly influenced by the orientation of its variable domains. We generated anti-c-Met scFv antibodies with two different domain orders (i.e., $V_L$-linker-$V_H$ and $V_H$-linker-$V_L$), expressed them in the cytoplasm of E. coli trx/gor deleted mutant, and compared their specific activities as well as their productivities. Productivity of total and functional anti-c-Met scFv with $V_H/V_L$ orientation was more than five times higher than that with $V_L/V_H$ format. Coexpression of DsbC enhanced the yield of soluble amounts of anti-c-Met scFv protein for both constructs. The purified scFv antibodies of the two different formats exhibited almost the same antigen-binding activities. We also compared the productivities and specific activities of anti-c-Met diabodies with $V_H/V_L$ or $V_L/V_H$ formats and obtained similar results to the case of scFv antibodies.

Keywords

References

  1. Arndt, M. A. E., J. Krauss, and S. M. Rybak. 2004. Antigen binding and stability properties of non-covalently linked anti- CD22 single-chain Fv dimers. FEBS Lett. 578: 257-261 https://doi.org/10.1016/j.febslet.2004.11.011
  2. Baneyx, F. 1999. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10: 411-421 https://doi.org/10.1016/S0958-1669(99)00003-8
  3. Bessette, P. H., F. Aslund, J. Beckwith, and G. Georgiou. 1999. Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc. Natl. Acad. Sci. 96: 13703- 13708
  4. Fernandez, L. A. 2004. Prokaryotic expression of antibodies and affibodies. Curr. Opin. Biotechnol. 15: 364-373 https://doi.org/10.1016/j.copbio.2004.06.004
  5. Hamilton, S., J. Odili, O. Gundogdu, G. D. Wilson, and J. Kupsch. 2001. Improved production by domain inversion of single-chain Fv antibody fragment against high molecular weight proteoglycan for the radioimmunotargeting of melanoma. Hybrid. Hybridomics 20: 351-360 https://doi.org/10.1089/15368590152740752
  6. Heo, M., S. Kim, S. Kim, Y. Kim, J. Chung, M. Oh, and S. Lee. 2006. Functional expression of single-chain variable fragment antibody against c-Met in the cytoplasm of Escherichia coli. Protein Expr. Purif. 47: 203-209 https://doi.org/10.1016/j.pep.2005.12.003
  7. Hudson, P. J. and C. Souriau. 2003. Engineered antibodies. Nat. Med. 9: 129-134 https://doi.org/10.1038/nm0103-129
  8. Jiang, W. G., T. A. Martin, C. Parr, G. Davies, K. Matsumoto, and T. Nakamura. 2005. Hepatocyte growth factor, its receptor, and their potential value in cancer therapies. Crit. Rev. Oncol. Hematol. 53: 35-69 https://doi.org/10.1016/j.critrevonc.2004.09.004
  9. Lee, S., J. Lee, J. Yi, and B. Kim. 2002. Production of cytidine 5-monophosphate N-acetylneuraminic acid using recombinant E. coli as a biocatalyst. Biotechnol. Bioeng. 80: 516-524 https://doi.org/10.1002/bit.10398
  10. Levy, R., R. Weiss, G. Chen, B. L. Iverson, and G. Georgiou. 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: 338-347 https://doi.org/10.1006/prep.2001.1520
  11. Lu, D., X. Jimenez, L. Witte, and Z. Zhu. 2004. The effect of variable domain orientation and arrangement on the antigenbinding activity of a recombinant human bispecific diabody. Biochem. Biophys. Res. Commun. 318: 507-513 https://doi.org/10.1016/j.bbrc.2004.04.060
  12. Makrides, S. C. 1996. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60: 512-538
  13. Maulik, G., A. Shrikhande, T. Kijima, P. C. Ma, P. T. Morrison, and R. Salgia. 2002. Role of the hepatocyte growth factor, c- Met, in oncogenesis and potential for therapeutic inhibition. Cytokine Growth Factor Rev. 13: 41-59 https://doi.org/10.1016/S1359-6101(01)00029-6
  14. Tsumoto, K., Y. Nakaoki, Y. Ueda, K. Ogasahara, K. Yutani, K. Watanabe, and I. Kumagai. 1994. Effect of the order of antibody variable regions on the expression of the single-chain hyhel10 Fv fragment in E. coli and the thermodynamic analysis of its antigen-binding properties. Biochem. Biophys. Res. Commun. 201: 546-551 https://doi.org/10.1006/bbrc.1994.1736
  15. Yang, Z. Y., W. B. Shim, M. G.. Kim, K. H. Lee, K. S. Kim, K. Y. Kim, C. H. Kim, S. D. Ha, and D. H. Chung. 2007. Production and characterization of monoclonal and recombinant antibodies against antimicrobial sulfamethazine. J. Microbiol. Biotechnol. 17: 571-578
  16. Yi, K. S., J. Chung, K. Park, K. Kim, S. Im, C. Choi, M. Im, and U. Kim. 2004. Expression system for enhanced green fluorescence protein conjugated recombinant antibody fragment. Hybrid. Hybridomics 23: 279-286 https://doi.org/10.1089/hyb.2004.23.279
  17. Yoo, M., J. Won, Y. Lee, and M. Choe. 2006. Increase of spacer sequence yields higher dimer (Fab-Spacer-Toxin)2 formation. J. Microbiol. Biotechnol. 16: 1097-1103