Single-Chain Fv Fragment of Catalytic Antibody 4f4f with Glycosidase Activity: Design, Expression, and Purification

• Published in: Journal of microbiology and biotechnology Vol. 9; no. 3; pp. 376 - 380
• Main Authors: Jang, Chang-Hwan, Chung, Hyun-Ho, Yu, Jae-Hoon, Chang, Yung-Jin, Kim, Hyong-Bai, Paek, Se-Hwan, Shin, Dong-Hoon, Kim, Kyung-Hyun
• Format: Journal Article
• Language: Korean
• Published: 1999
• Subjects: expression; catalytic antibody; engineering; Single-chain Fv fragment
• ISSN: 1017-7825; 1738-8872

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.