Open AccessEffect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin
Author(s) -
Hong Yun Tham,
Adelene A-L Song,
Khatijah Yusoff,
Geok Hun Tan
Publication year - 2020
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/btn-2020-0034
Subject(s) - lysin , xhoi , bacteriophage , microbiology and biotechnology , escherichia coli , biology , recombinant dna , cloning (programming) , biochemistry , gene , computer science , bamhi , programming language
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 Bam HI/ Xho 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 Nco I/ Xho 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.