Rational design based on translation pausing theory significantly enhances the soluble expression and activity of multidomain anti-CD20 fab antibody sequences

The expression of IgG antibodies in Chinese hamster ovary (CHO) cells has been widely used, but their expression in Escherichia coli remains challenging. In antibody engineering and drug research, the expression and functional study of antibody fragments in E. coli is crucial for early development. However, these fragments often aggregate to form inclusion bodies or exhibit low solubility when expressed in the periplasm, making them difficult to obtain. In this study, we propose a strategy based on the theory of translational pausing, where mutations are introduced into the DNA sequence to alter the translation pausing sites without changing the amino acid sequence, thus promoting correct protein folding and improving the solubility of heterologous proteins in E. coli. We previously successfully optimized the antiviral protein cyanovirin-N (CVN) from cyanobacteria and epoxide hydrolase (EH-Ar) from Agrobacterium, and we attempted to express an anti-CD20 Fab antibody with a quaternary structure to verify whether the translational pausing technique can increase the solubility and expression of multidomain proteins. Compared with the wild-type sequence, the designed coding sequence exhibited greater soluble expression in E. coli, and the expressed Fab antibody retained its activity. This study further demonstrates the importance of translational pausing in the expression of heterologous proteins in bacterial hosts and provides a novel approach for soluble expression and rapid preparation of recombinant small-molecule antibodies.