Expression of soluble and functional single-chain antibody in Escherichia coli

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Expression of soluble and functional single-chain antibody in Escherichia coli
Sung Sup Park; Byung Rae Jin; Moon Hi Han; Hyo Jeong Hong
Bibliographic Citation
Molecules and Cells, vol. 4, pp. 413-417
Publication Year
We investigated the growth conditions for the production of soluble and functional single-chain antibody (ScFv) accumulated in the periplasm of E. coli, purified the periplasmic soluble ScFv protein and confirmed that the ScFv in functional. The ScFv (VH-linker-VL) gene fused to pelB signal sequence, which is specific for the pre-S2 surface antigen of hepatitis B virus (HBV), was synthesized by PCR and linked to strong T7 promoter to construct expression plasmid pTVS2. The resulting plasmid was expressed in E. coli BL21(DE3) at 37 ˚C, 30 ˚C or 24 ˚C and it was found that the growth of the recombinant cells was more inhibited as the induction remperature was raised. Accordingly, synthesis of the ScFv protein was induced at 24 ˚C for 5 h and the location of the expressed ScFv in the induced cells was analyzed by fractionation of the total protein extract, SDS-PAGE and Western blot, suggesting that transport of the ScFv is precessed normally in the cells but the folding is limiting in the periplasm and thus only a certain fraction of the transported proteins are assembled by periplasmic folding. Periplasmic souble ScFv protein could be purified to homogeneity by an antigen affinity chromatography with a yield of 0.8 mg/l culture and also as shown to bind specifically to the pre-S2 peptide antigen with amd affinity of 5 X 10 7 M-1, about 1/7 that of the parental murine monoclonal antibody, suggesting that it is functional.
Korea Soc-Assoc-Inst
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Aging Convergence Research Center > 1. Journal Articles
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