Improved soluble expression of a single-chain antibody fragment in E. coli for targeting CA125 in epithelial ovarian cancer

•We report enhanced soluble expression of the anti-CA125 B43.13 scFv in E. coli.•The anti-CA125 scFv gene was codon-optimized for expression in E. coli.•A 3.8–14 fold increase in soluble expression was obtained by codon optimization.•All scFv variants expressed and purified were immunoreactive to CA...

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Bibliographic Details
Published in:Protein expression and purification 2014-10, Vol.102, p.27-37
Main Authors: Sharma, Sai Kiran, Suresh, Mavanur R., Wuest, Frank R.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Base Sequence
CA-125 Antigen - immunology
CA125
Carcinoma, Ovarian Epithelial
Cell Line, Tumor
Cloning, Molecular - methods
Codon - genetics
Codon optimization
Epithelial ovarian cancer
Escherichia coli - genetics
Humans
Immunoglobulin Variable Region
Inter-domain peptide linker
Membrane Proteins - immunology
Molecular Sequence Data
Neoplasms, Glandular and Epithelial - immunology
Ovarian Neoplasms - immunology
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - immunology
Single-Chain Antibodies - chemistry
Single-Chain Antibodies - genetics
Single-Chain Antibodies - immunology
Single-chain Fragment variable (scFv)
Solubility
Soluble expression
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Summary:•We report enhanced soluble expression of the anti-CA125 B43.13 scFv in E. coli.•The anti-CA125 scFv gene was codon-optimized for expression in E. coli.•A 3.8–14 fold increase in soluble expression was obtained by codon optimization.•All scFv variants expressed and purified were immunoreactive to CA125. Production of antibody fragments in heterologous hosts such as Escherichia coli 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.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2014.07.007