Enhanced periplasmic expression of high affinity humanized scFv against Hepatitis B surface antigen by codon optimization.

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.