An effective strategy for soluble bovine enterokinase expression in Escherichia coli.

Bovine enterokinase light chain (bEkL) is a serine protease, widely used for the specific cleavage of affinity tags from various recombinant proteins. However, getting soluble expression in Escherichia coli is a challenging task given the presence of multiple cysteines and four disulfide bonds. Strategies that have only been partially successful involve mutating the gene or covalent attachment of solubility tags. We demonstrate a simpler and more efficient production method that combines different strategies like co-expressing the GroES-GroEL chaperone in E. coli SHuffle cells, lowering temperatures to 18 °C post-induction, and ensuring the sufficient accumulation of GroES-GroEL in the cytoplasm before inducing the bEkL gene. A trade-off exists between producing too little GroES-GroEL and promoting inclusion body formation (of bEkL) or expressing too much GroES-GroEL thereby reducing bEkL yields. This optimum level was determined by varying the time difference between the two inductions, and the best results obtained when the co-expressed pGro7 plasmid was induced 2 h before bEkL induction, thus avoiding inclusion body formation. Interestingly, when we delayed the induction of GroES-GroEL to an OD