title: Overexpression of DsbC and DsbG markedly improves soluble and functional expression of single-chain Fv antibodies in Escherichia coli.
abstract: Single-chain Fv antibodies (scFv), a group of reconstructed molecules with several disulfide bonds, are prone to aggregate as inclusion bodies, the insoluble species of natural proteins, when expressed in Escherichia coli, especially at high level. Recovery of functionally active products from inclusion bodies is onerous and ineffective. We have increased the soluble and functional scFv yields by fusing either DsbC or DsbG, two E. coli disulfide isomerases with general chaperone function, to scFvs. Compared to the totally insoluble inclusion bodies of scFvs expressed separately, more than half of each fusion protein DsbC-scFv or DsbG-scFv was soluble, according to SDS-PAGE analysis. The more effective solubility was obtained when the fused protein DsbG-scFv was co-expressed simultaneously with DsbC under the same promoter. Under this condition, the soluble portion of DsbG-scFv increased from about 50% to 90% measured by scanning SDS-PAGE gel. Co-expression of DsbC can change fusion protein CBD-scFv from totally insoluble when expressed in E. coli separately to a considerable portion of soluble CBD-scFv. Antigen-binding activity assay showed that scFvs retained full affinity to specific antigens. We also determined that general molecular chaperones GroEL and GroES had no effects on the solubility of scFvs when co-expressed with scFv in E. coli. We propose that the correct formation of disulfide bonds in scFvs is the crucial factor responsible for solubility of scFvs.
authors.affiliation: Group 102, Institute of Genetics and Developmental Biology, Academia Sinica, Beijing 100101, China.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Zhang
authors.forename: Zhi-Hua
authors.initials: ZH
authors.lastname: Li
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.forename: Min
authors.initials: M
authors.lastname: Fang
authors.forename: Chang-Cheng
authors.initials: CC
authors.lastname: Yin
authors.forename: Zhi-Yong
authors.initials: ZY
authors.lastname: Zhou
authors.forename: Qing
authors.initials: Q
authors.lastname: Lin
authors.forename: Hua-Liang
authors.initials: HL
authors.lastname: Huang
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D010957:Plasmids; D019704:Protein Disulfide-Isomerases / Q000235:genetics*; D012995:Solubility
chemical_list: D017931:DNA Primers; D007128:Immunoglobulin Fragments; D019704:Protein Disulfide-Isomerases