title: Combination of Dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain Fv in Escherichia coli.
abstract: 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 be applicable for high-yield soluble expression of proteins with multiple disulfide bonds.
authors.affiliation: Department of Biotechnology, Graduate school of Engineering, Osaka University, Suita, Osaka, Japan.
authors.forename: Duanpen
authors.initials: D
authors.lastname: Sandee
authors.forename: Sumalee
authors.initials: S
authors.lastname: Tungpradabkul
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Kurokawa
authors.forename: Kiichi
authors.initials: K
authors.lastname: Fukui
authors.forename: Masahiro
authors.initials: M
authors.lastname: Takagi
mesh_terms: D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D001622:Betaine / Q000494:pharmacology; D001667:Binding, Competitive; D049109:Cell Proliferation; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism*; D005822:Genetic Vectors; D006131:Growth Inhibitors / Q000494:pharmacology; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000187:drug effects / Q000502:physiology; D057127:Single-Chain Antibodies; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility; D013012:Sorbitol / Q000494:pharmacology*; D013395:Sucrose / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; C079503:DsbB protein, Bacteria; D006131:Growth Inhibitors; C478634:Hep27scFv; D007128:Immunoglobulin Fragments; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D057127:Single-Chain Antibodies; D001622:Betaine; D012965:Sodium Chloride; D013012:Sorbitol; D013395:Sucrose