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KMID : 0545120080180061186
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Journal of Microbiology and Biotechnology
2008 Volume.18 No. 6 p.1186 ~ p.1190
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Improving the Productivity of Single-Chain Fv Antibody Against c-Met by Rearranging the Order of its Variable Domains
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Kim Yu-Jin
Neelamegam Rameshkumar
Heo Mi-Ae
Edwardraja Selvakumar
Paik Hyun-Jong
Lee Sun-Gu
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Abstract
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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., VL-linker-VH and VHlinker- VL), 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 VH/VL orientation was more than five times higher than that with VL/VH 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 VH/VL or VL/VH formats and obtained similar results to the case of scFv antibodies.
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KEYWORD
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Anti-c-Met scFv, variable domain, domain order, diabody
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