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


Citation

Tham, Hong Y. and Song, Adelene Ai Lian and Yusoff, Khatijah and Tan, Geok Hun (2020) Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin. BioTechniques, 69 (3). 161 - 170. ISSN 0736-6205; ESSN: 1940-9818

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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Additional Metadata

Item Type: Article
Divisions: Faculty of Agriculture
Faculty of Biotechnology and Biomolecular Sciences
Institute of Bioscience
DOI Number: https://doi.org/10.2144/btn-2020-0034
Publisher: Future Science
Keywords: Antimicrobial activity; Endolysin; Inclusion body; MRSA; pET28; Protein expression
Depositing User: Ms. Nuraida Ibrahim
Date Deposited: 22 Dec 2021 02:03
Last Modified: 22 Dec 2021 02:03
Altmetrics: http://www.altmetric.com/details.php?domain=psasir.upm.edu.my&doi=10.2144/btn-2020-0034
URI: http://psasir.upm.edu.my/id/eprint/88588
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