Combination of Dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain Fv in Escherichia coli
Many eukaryotic proteins have been produced successfully in Escherichia coli. However, not every gene can be expressed efficiently in this organism. Most proteins, especially those with multiple disulfide bonds, have been shown to form insoluble protein or inclusion body in E. coli. An inactive form...
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th-mahidol.163022018-06-21T15:15:25Z Combination of Dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain Fv in Escherichia coli Duanpen Sandee Sumalee Tungpradabkul Yoichi Kurokawa Kiichi Fukui Masahiro Takagi Osaka University Mahidol University Fukui Prefectural University Japan Advanced Institute of Science and Technology Biochemistry, Genetics and Molecular Biology Immunology and Microbiology Many eukaryotic proteins have been produced successfully in Escherichia coli. However, not every gene can be expressed efficiently in this organism. Most proteins, especially those with multiple disulfide bonds, have been shown to form insoluble protein or inclusion body in E. coli. An inactive form of protein would require an in vitro refolding step to regain biological functions. In this study, we described the system for soluble expression of a single-chain variable fragment (scFv) against hepatocellular carcinoma (Hep27scFv) by coexpressing Dsb protein and enhancing with medium additives. The results revealed that overexpression of DsbABCD protein showed marked effect on the soluble production of Hep27scFv, presumably facilitating correct folding. The optimal condition for soluble scFv expression could be obtained by adding 0.5M sorbitol to the culture medium. The competitive enzyme-linked immunosorbent assay (ELISA) indicated that soluble scFv expressed by our method retains binding activity toward the same epitope on a hepatocellular carcinoma cell line (HCC-S102) recognized by intact antibody (Ab) (Hep27 Mab). Here, we report an effective method for soluble expression of scFv in E. coli by the Dsb coexpression system with the addition of sorbitol medium additive. This method might beapplicableforhigh-yield solubleexpression of proteins with multiple disulfide bonds. © 2005 Wiley Periodicals, Inc. 2018-06-21T08:08:25Z 2018-06-21T08:08:25Z 2005-08-20 Article Biotechnology and Bioengineering. Vol.91, No.4 (2005), 418-424 10.1002/bit.20524 00063592 2-s2.0-23244448782 https://repository.li.mahidol.ac.th/handle/123456789/16302 Mahidol University SCOPUS https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=23244448782&origin=inward |
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Biochemistry, Genetics and Molecular Biology Immunology and Microbiology Duanpen Sandee Sumalee Tungpradabkul Yoichi Kurokawa Kiichi Fukui Masahiro Takagi Combination of Dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain Fv in Escherichia coli |
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Many eukaryotic proteins have been produced successfully in Escherichia coli. However, not every gene can be expressed efficiently in this organism. Most proteins, especially those with multiple disulfide bonds, have been shown to form insoluble protein or inclusion body in E. coli. An inactive form of protein would require an in vitro refolding step to regain biological functions. In this study, we described the system for soluble expression of a single-chain variable fragment (scFv) against hepatocellular carcinoma (Hep27scFv) by coexpressing Dsb protein and enhancing with medium additives. The results revealed that overexpression of DsbABCD protein showed marked effect on the soluble production of Hep27scFv, presumably facilitating correct folding. The optimal condition for soluble scFv expression could be obtained by adding 0.5M sorbitol to the culture medium. The competitive enzyme-linked immunosorbent assay (ELISA) indicated that soluble scFv expressed by our method retains binding activity toward the same epitope on a hepatocellular carcinoma cell line (HCC-S102) recognized by intact antibody (Ab) (Hep27 Mab). Here, we report an effective method for soluble expression of scFv in E. coli by the Dsb coexpression system with the addition of sorbitol medium additive. This method might beapplicableforhigh-yield solubleexpression of proteins with multiple disulfide bonds. © 2005 Wiley Periodicals, Inc. |
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Osaka University |
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Osaka University Duanpen Sandee Sumalee Tungpradabkul Yoichi Kurokawa Kiichi Fukui Masahiro Takagi |
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Article |
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Duanpen Sandee Sumalee Tungpradabkul Yoichi Kurokawa Kiichi Fukui Masahiro Takagi |
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Duanpen Sandee |
title |
Combination of Dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain Fv in Escherichia coli |
title_short |
Combination of Dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain Fv in Escherichia coli |
title_full |
Combination of Dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain Fv in Escherichia coli |
title_fullStr |
Combination of Dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain Fv in Escherichia coli |
title_full_unstemmed |
Combination of Dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain Fv in Escherichia coli |
title_sort |
combination of dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain fv in escherichia coli |
publishDate |
2018 |
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https://repository.li.mahidol.ac.th/handle/123456789/16302 |
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1763495891981828096 |