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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| Format: | Article |
| Language: | English |
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Future Science
2020
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| Online Access: | http://psasir.upm.edu.my/id/eprint/88588/ http://psasir.upm.edu.my/id/eprint/88588/1/ABSTRACT.pdf |
| _version_ | 1848860650855464960 |
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| author | Tham, Hong Y. Song, Adelene Ai Lian Yusoff, Khatijah Tan, Geok Hun |
| author_facet | Tham, Hong Y. Song, Adelene Ai Lian Yusoff, Khatijah Tan, Geok Hun |
| author_sort | Tham, Hong Y. |
| building | UPM Institutional Repository |
| collection | Online Access |
| 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. |
| first_indexed | 2025-11-15T12:48:37Z |
| format | Article |
| id | upm-88588 |
| institution | Universiti Putra Malaysia |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-15T12:48:37Z |
| publishDate | 2020 |
| publisher | Future Science |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | upm-885882021-12-22T02:03:19Z 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 |
| 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 |
| title | 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_short | 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 |
| url | http://psasir.upm.edu.my/id/eprint/88588/ http://psasir.upm.edu.my/id/eprint/88588/ http://psasir.upm.edu.my/id/eprint/88588/ http://psasir.upm.edu.my/id/eprint/88588/1/ABSTRACT.pdf |