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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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Official URL: http://dx.doi.org/10.1039/c8ra06237d

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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
Subjects: Q Science > QR Microbiology > QR355 Virology
Divisions: Faculty of Science, Engineering and Medicine > 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:
DateEvent
25 September 2018Available
14 September 2018Accepted
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:
Project/Grant IDRIOXX Funder NameFunder ID
Sir Henry Dale FellowshipWellcome Trusthttp://dx.doi.org/10.13039/100010269
104193/Z/14/Z[RS] Royal Societyhttp://dx.doi.org/10.13039/501100000288
201442/Z/16/ZWellcome Trusthttp://dx.doi.org/10.13039/100010269
RG120405[RS] Royal Societyhttp://dx.doi.org/10.13039/501100000288

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