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  • Title: Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin.
    Author: Tham HY, Song AA, Yusoff K, Tan GH.
    Journal: Biotechniques; 2020 Sep; 69(3):161-170. PubMed ID: 32787565.
    Abstract:
    Endolysins have been studied intensively as an alternative to antibiotics. In this study, endolysin derived from a phage which infects methicillin-resistant Staphylococcus aureus (MRSA) was cloned and expressed in Escherichia coli pET28a. Initially, the endolysin was cloned using BamHI/XhoI, 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 NcoI/XhoI 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.
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