Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin
| Autor: | Hong Y Tham, Geok Hun Tan, Khatijah Yusoff, Adelene A-L Song |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Methicillin-Resistant Staphylococcus aureus
XhoI Lysin medicine.disease_cause General Biochemistry Genetics and Molecular Biology Inclusion bodies law.invention Microbiology 03 medical and health sciences law Endopeptidases Escherichia coli medicine Humans Bacteriophages 030304 developmental biology Cloning 0303 health sciences biology 030306 microbiology Chemistry Recombinant Proteins Gene Expression Regulation Staphylococcus aureus biology.protein Recombinant DNA BamHI Biotechnology |
| Zdroj: | BioTechniques. 69:161-170 |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/btn-2020-0034 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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