Directed evolution for soluble and active periplasmic expression of bovine enterokinase in Escherichia coli.

ABSTRACT: Bovine enterokinase light chain (EK_L) is an industrially useful protease for accurate removal of affinity-purification tags from high-value biopharmaceuticals. However, recombinant expression in Escherichia coli produces insoluble inclusion bodies, requiring solubilisation, refolding, and autocatalytic activation to recover functional enzyme. Error-prone PCR and DNA shuffling of the EK_L gene, T7 promoter, lac operon, ribosome binding site, and pelB leader sequence, yielded 321 unique variants after screening ~ 6500 colonies. The best variants had > 11,000-fold increased total activity in lysates, producing soluble enzyme that no longer needed refolding. Further characterisation identified the factors that improved total activity from an inactive and insoluble starting

SUBMITTER: Lee W 

PROVIDER: S-EPMC9587228 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature