Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin

Endolysins have been studied intensively as an alternative to antibiotics. In this study, endolysin derived from a phage which infects methicillin-resistant (MRSA) was cloned and expressed in pET28a. Initially, the endolysin was cloned using HI/ I, resulting in expression of a recombinant endolysin which was expressed in inclusion bodies. While solubilization was successful, the protein remained nonfunctional. Recloning the endolysin using I/ I resulted in expression of soluble and functional proteins at 18°C. The endolysin was able to form halo zones on MRSA plates and showed a reduction in turbidity of MRSA growth. Therefore, cloning strategies should be chosen carefully even in an established expression system as they could greatly affect the functionality of the expressed protein. Cloning of endolysin in pET28a using HI and I resulted in insoluble and nonfunctional recombinant protein. Changing HI to I while maintaining I resulted in soluble and functional proteins expressed at 18°C. This could be due to too many additional amino acids added from the pET28a plasmid in the recombinant protein when cloning using HI, the N-terminus His-tag causing improper folding of the protein, or a combination of both. Cloning of the endolysin using I- I, which had the His-tag at the C-terminus and fewer additional amino acids introduced during cloning, resulted in successful functional protein expression.