An industrial available platform for high-yield, plasmid-free recombinant protein production in E. coli based on...

Protein production is a cornerstone of biotechnology, and the cleavable self- aggregating tag (cSAT) scheme has been developed for column-free purification of recombinant proteins. We present an advanced cSAT (acSAT) scheme for high-yield recombinant protein production in E. coli. The acSAT scheme integrates a linker between the intein and target protein, reducing premature cleavage and enhancing protein yield. We screened ten linkers, optimizing the cleavage efficiency and yield of model proteins such as collagen type III (COL-III), fibronectin (FN), and fusion proteins (FP). Further optimization using dual-linkers resulted in improved yields of truncated COL-III (tCOL-III), with dual-linker L13 increasing the yield of tCOL-III by 119 %. Additionally, we identified a high-performing neutral genomic integration site near the oriC of E. coli with integration efficiency nearly 100 %, enabling plasmid-free, antibiotic-free expression systems for large-scale production. In 5-L fed-batch fermentation, acSAT scheme yielded up to 1.51 g/L tCOL-III, which was 27.2 times higher than that of shake flask cultures. This platform offers a cost-effective, scalable solution for industrial recombinant protein production.