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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 Staphylococcus aureus (MRSA) was cloned and expressed in Escherichia coli pET28a. Initially, the endolysin was cloned using BamHI/XhoI, resulti...

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Autori principali: Tham, Hong Y., Song, Adelene Ai Lian, Yusoff, Khatijah, Tan, Geok Hun
Natura: Articolo
Lingua:English
Pubblicazione: Future Science 2020
Accesso online:http://psasir.upm.edu.my/id/eprint/88588/1/ABSTRACT.pdf
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spelling oai:psasir.upm.edu.my:88588 http://psasir.upm.edu.my/id/eprint/88588/ Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin Tham, Hong Y. Song, Adelene Ai Lian Yusoff, Khatijah Tan, Geok Hun 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. Future Science 2020 Article PeerReviewed text en http://psasir.upm.edu.my/id/eprint/88588/1/ABSTRACT.pdf 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 https://www.future-science.com/doi/10.2144/btn-2020-0034 10.2144/btn-2020-0034
institution UPM IR
collection UPM IR
language English
description 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.
format Article
author Tham, Hong Y.
Song, Adelene Ai Lian
Yusoff, Khatijah
Tan, Geok Hun
spellingShingle Tham, Hong Y.
Song, Adelene Ai Lian
Yusoff, Khatijah
Tan, Geok Hun
Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin
author_facet Tham, Hong Y.
Song, Adelene Ai Lian
Yusoff, Khatijah
Tan, Geok Hun
author_sort Tham, Hong Y.
title Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin
title_short Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin
title_full Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin
title_fullStr Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin
title_full_unstemmed Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin
title_sort effect of different cloning strategies in pet-28a on solubility and functionality of a staphylococcal phage endolysin
publisher Future Science
publishDate 2020
url http://psasir.upm.edu.my/id/eprint/88588/1/ABSTRACT.pdf
_version_ 1819300267518066688
score 13.4562235

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