title: Improved soluble expression of a single-chain antibody fragment in E. coli for targeting CA125 in epithelial ovarian cancer.
abstract: Production of antibody fragments in heterologous hosts such as Escherichiacoli provides a unique and cost-effective method to develop engineered vectors for tumor targeting. A single-chain Fragment variable (scFv) of the murine monoclonal antibody MAb-B43.13 targeting CA125 in epithelial ovarian cancer was previously developed, expressed, purified and proposed as a functional targeting entity for biomedical applications. However, the yields from its soluble expression in heterologous systems were very low for any practical use in preclinical translational research; leave alone the defeated objective of convenient and cost-effective production. In the present work, the anti-CA125 scFv gene was re-organized and sub-cloned into pET-22b(+) vector to be in frame with the pelB leader peptide for periplasmic localization and C-terminal hexa-histidine tag to facilitate downstream purification. Six variants of the scFv were constructed to investigate the impact of variable domain orientations, inter-domain peptide linker sequences and codon optimization on the soluble expression of the scFv using Rosetta 2(DE3) as the E. coli host supplemented with tRNAs for rare codons. Expression in shake flask cultures under the control of an inducible T7 promoter and subsequent purification by cobalt based immobilized metal affinity chromatography yielded differential amounts of high purity scFv for all constructs. Here, we report up to 14-fold increase in the soluble expression of the scFv primarily as a result of codon optimization with minor effects from inter-domain peptide linkers and variable domain orientation in the anti-CA125 scFv molecule. All the scFv constructs expressed and purified were found to be immunoreactive for in vitro targeting of CA125 antigen.
authors: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada; Department of Oncology, University of Alberta, Edmonton, Canada.
authors: Sai Kiran
authors: SK
authors: Sharma
authors: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada.
authors: Mavanur R
authors: MR
authors: Suresh
authors: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada; Department of Oncology, University of Alberta, Edmonton, Canada. Electronic address: wuest@ualberta.ca.
authors: Frank R
authors: FR
authors: Wuest
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D018394:CA-125 Antigen / Q000276:immunology*; D000077216:Carcinoma, Ovarian Epithelial; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins / Q000276:immunology*; D008969:Molecular Sequence Data; D009375:Neoplasms, Glandular and Epithelial / Q000276:immunology*; D010051:Ovarian Neoplasms / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D012995:Solubility
chemical_list: D018394:CA-125 Antigen; D003062:Codon; D007135:Immunoglobulin Variable Region; C451759:MUC16 protein, human; D008565:Membrane Proteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
keywords: CA125; Codon optimization; Epithelial ovarian cancer; Inter-domain peptide linker; Single-chain Fragment variable (scFv); Soluble expression