Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Single-Chain Fv Fragment of Catalytic Antibody 4f4f with Glycosidase Activity: Design, Expression, and Purification

  • Jang, Chang-Hwan (Graduate School of Biotechnology, Korea University) ;
  • Chung, Hyun-Ho (Biotech Research Institute, LG Chemical Ltd./Research Park) ;
  • Yu, Jae-Hoon (Division of Applied Science, Korea Institute of Science and Technology) ;
  • Chang, Yung-Jin (Department of Agricultural Chemistry, Seoul National University) ;
  • Kim, Hyong-Bai (Graduate School of Biotechnology, Korea University) ;
  • Paek, Se-Hwan (Graduate School of Biotechnology, Korea University) ;
  • Shin, Dong-Hoon (Graduate School of Biotechnology, Korea University) ;
  • Kim, Kyung-Hyun (Graduate School of Biotechnology, Korea University)
  • Published : 1999.06.01
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Abstract

Constructs, encoding a single-chain variable fragment of a catalytic antibody 4f4f (scFv-4f4f) with glycosidase activity, were made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding a linker (GGGGS). Using three different plasmid systems, single-chain antibodies were expressed separately in Escherichia coli, demonstrating significant differences in the expression level and amounts in soluble form of the recombinant protein. The protein expression from pET3a-scFv-4f4f was up to 20% of the total soluble proteins and, more importantly, the proteins were mostly found in a soluble form. An SDS-PAGE analysis of the purified single-chain proteins, yielding higher than 5mg from a 1-1 culture, showed a single band corresponding to its molecular weight of 29,100. A preliminary study shows that the expressed scFv-4f4f is catalytically active. The catalytic parameters for the hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside by scFv-4f4f are being investigated.

Keywords

References

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