Extracellular production of Ulp1403-621 in leaky E. coli and its application in antimicrobial peptide production

Small ubiquitin-like modifier (SUMO) tag is widely used to promote soluble expression of exogenous proteins, which can then be cleaved by ubiquitin-like protease 1 (Ulp1) to obtain interested protein. But the application of Ulp1 in large-scale recombinant protein production is limited by complicated purification procedures and high cost. In this study, we describe an efficient and simple method of extracellular production of Ulp1 403-621 using a leaky Escherichia coli BL21(DE3), engineered by deleting the peptidoglycan-associated outer membrane lipoprotein ( pal ) gene. Ulp1 403-621 was successfully leaked into extracellular supernatant by the BL21(DE3)-Δ pal strain after IPTG induction. The addition of 1% glycine increased the extracellular production of Ulp1 403-621 approximately four fold. Moreover, extracellular Ulp1 403-621 without purification had high activities for cleaving SUMO fusion proteins, and antimicrobial peptide pBD2 obtained after cleavage can inhibit the growth of Staphylococcus aureus . The specific activity of extracellular Ulp1 403-621 containing 1 mM EDTA and 8 mM DTT reached 2.0 × 10 6 U/L. Another commonly used protease, human rhinovirus 3C protease, was also successfully secreted by leaky E. coli strains. In conclusion, extracellular production of tool enzymes is an attractive way for producing large-scale active recombinant proteins at a lower cost for pharmaceutical, industrial, and biotechnological applications. Key points • First report of extracellular production of Ulp1 403-621 in leaky Escherichia coli BL21(DE3) strain. • One percent glycine addition into cultivation medium increased the extracellular production of Ulp1 403-621 approximately four fold. • The specific activity of extracellular Ulp1 403-621 produced in this study reached 2.0 × 10 6 U/L.