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KMID : 0578319940040040413
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Molecules and Cells
1994 Volume.4 No. 4 p.413 ~ p.417
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Expression of Soluble and Functional Single-Chain Antibody in Escherichia coli
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Park, Sung Sup
Jin, Byung Rae/Han, Moon Hi/Hong, Hyo Jeong
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Abstract
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We investigated the growth conditions for the production of soluble and functional single-chain antibody (ScFv) accumulated in the periplasm of E coh, purified the periplasmic soluble ScFv protein and confirmed that the ScFv is functional. The ScFv (VH-Gnker-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 colt B121(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 temperature 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, SOS-PAGE and Western blotting. The results showed that the ScFv was highly expressed and was present mostly in the periplasmic insoluble fraction but the periplasmic soluble ScFv was detected on the Western blot, suggesting that transport of the ScFv is processed 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 soluble ScFv protein could be purified to homogeneity by an antigen affinity chromatography with a yield of 0.8 mg/I culture and also was shown to bind specifically to the pre-S2 peptide antigen with an affinity of 5 X 10´ M´, about 1/7 that of the parental murine monoclonal antibody, suggesting that it is functional.
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