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KMID : 0545119990090030376
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Journal of Microbiology and Biotechnology
1999 Volume.9 No. 3 p.376 ~ p.380
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Single-Chain Fv Fragment of Catalytic Antibody 4f4f with Glycosidase Activity
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Jang, Chang Hwan
Chung, Hyun Ho/Yu, Jae Hoon/Chang, Yung Jin/Kim, Hyong Bai/Paek, Se Hwan/Shin, Dong Hoon
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Abstract
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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 singlechain proteins, yielding higher than 5㎎ 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-β-D-glucopyranoside by scFv4f4f are being investigated.
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