title: Enhanced periplasmic expression of high affinity humanized scFv against Hepatitis B surface antigen by codon optimization.
abstract: Production of properly folded, functional recombinant antibodies in a prokaryotic system is governed by multiple factors like codon usage, plasmid copy number, upstream elements such as leader sequence, mRNA stability and presence of tightly controlled promoters. Here we present a strategy for enhanced production of the functional scFv in Escherichia coli by codon optimization. We have previously reported the generation of humanized scFv form of a potentially neutralizing mouse monoclonal antibody (5S) to the Hepatitis B surface antigen. However, the expression level of 5S-scFv in E. coli was fairly low which was possibly due to the presence of rare codons. In the native 5S-scFv gene, almost 58% of codons showed poor codon bias with varying degrees of rare occurrence in the E. coli genes. We therefore designed a synthetic gene encoding the 5S-scFv protein by using E. coli preferred codon usage. The codon-optimized scFv gene was further cloned into a T7 expression system with a C-terminus His-tag and expressed as a soluble protein mainly in the periplasm. The scFv was both purified by IMAC and detected on Western blot with this His-tag. Using the codon optimization strategy, we were able to achieve a more than 100-fold increased periplasmic expression of soluble scFv. Further, the purified scFv was stable and retained its antigen-binding affinity and epitope specificity. Interestingly, based on secondary structure prediction, we observed that the mRNA secondary structure, including that of the 5'-end, may not have a significant role in the increased expression of this optimized gene.
authors.affiliation: Department of Biochemistry, All India Institute of Medial Sciences, New Delhi, India. ashutosht@hotmail.com
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Tiwari
authors.forename: Anurag
authors.initials: A
authors.lastname: Sankhyan
authors.forename: Navin
authors.initials: N
authors.lastname: Khanna
authors.forename: Subrata
authors.initials: S
authors.lastname: Sinha
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003062:Codon; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D019897:Periplasm / Q000378:metabolism; D015202:Protein Engineering; D057127:Single-Chain Antibodies / Q000235:genetics*
chemical_list: D003062:Codon; D006514:Hepatitis B Surface Antigens; D007128:Immunoglobulin Fragments; D057127:Single-Chain Antibodies