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A GFP-strategy for efficient recombinant protein overexpression and purification in Mycobacterium smegmatis
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Radhakrishnan, Anjana, Furze, Christopher M., Ahangar, Mohd Syed and Fullam, Elizabeth (2018) A GFP-strategy for efficient recombinant protein overexpression and purification in Mycobacterium smegmatis. RSC Advances , 8 (58). pp. 33087-33095. doi:10.1039/c8ra06237d
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WRAP-GFP-strategy-efficient-recombinant-protein-overexpression-purification-Mycobacterium-smegmatis-Fullam-2018.pdf - Published Version - Requires a PDF viewer. Available under License Creative Commons Attribution 4.0. Download (916Kb) | Preview |
Official URL: http://dx.doi.org/10.1039/c8ra06237d
Abstract
One of the major obstacles to obtaining a complete structural and functional understanding of proteins encoded by the Mycobacterium tuberculosis (Mtb) pathogen is due to significant difficulties in producing recombinant mycobacterial proteins. Recent advances that have utilised the closely related Mycobacterium smegmatis species as a native host have been effective. Here we have developed a method for the rapid screening of both protein production and purification strategies of mycobacterial proteins in whole M. smegmatis cells following green fluorescent protein (GFP) fluorescence as an indicator. We have adapted the inducible T7-promoter based pYUB1062 shuttle vector by the addition of a tobacco etch virus (TEV) cleavable C-terminal GFP enabling the target protein to be produced as a GFP-fusion with a poly-histidine tag for affinity purification. We illustrate the advantages of a fluorescent monitoring approach with the production and purification of the mycobacterial N-acetylglucosamine-6-phosphate deacetylase (NagA)-GFP fusion protein. The GFP system described here will accelerate the production of mycobacterial proteins that can be used to understand the molecular mechanisms of Mtb proteins and facilitate drug discovery efforts.
| Item Type: | Journal Article | |||||||||||||||
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| Subjects: | Q Science > QR Microbiology > QR355 Virology | |||||||||||||||
| Divisions: | Faculty of Science > Life Sciences (2010- ) | |||||||||||||||
| Library of Congress Subject Headings (LCSH): | Mycobacterium tuberculosis, Tuberculosis -- Molecular aspects, Mycobacteria, Green fluorescent protein, Recombinant proteins, Overexpression (Genetics) | |||||||||||||||
| Journal or Publication Title: | RSC Advances | |||||||||||||||
| Publisher: | Royal Society of Chemistry | |||||||||||||||
| ISSN: | 2046-2069 | |||||||||||||||
| Official Date: | 25 September 2018 | |||||||||||||||
| Dates: |
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| Date of first compliant deposit: | 25 September 2018 | |||||||||||||||
| Volume: | 8 | |||||||||||||||
| Number: | 58 | |||||||||||||||
| Page Range: | pp. 33087-33095 | |||||||||||||||
| DOI: | 10.1039/c8ra06237d | |||||||||||||||
| Status: | Peer Reviewed | |||||||||||||||
| Publication Status: | Published | |||||||||||||||
| Access rights to Published version: | Open Access | |||||||||||||||
| RIOXX Funder/Project Grant: |
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