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.
Highlights
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of human mortality from an infectious agent
In order to construct a uorescent indicator system to monitor recombinant protein expression in M. smegmatis mc24517 we selected the pYUB1062 shuttle vector,[21] which can replicate in both E. coli and M. smegmatis, for incorporation of a C-terminal fused green fluorescent protein (GFP)-His6-tag to the target protein
Activity of Nacetylglucosamine-6-phosphate deacetylase (NagA)-GFP-His[6] To con rm that production of protein fused with a C-terminal GFP reporter tag is not detrimental to activity of the protein we examined the catalytic deacetylation of N-acetylglucosamine6-phosphate (GlcNAc6P) substrate by both the NagA-GFP-His[6] fusion and the cleaved NagA enzymes using a uorescence assay.[19]
Summary
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of human mortality from an infectious agent. In 2016, 1.7 million deaths and 10.4 million new cases of TB were reported by the WHO.[1] Whilst TB can be treated, the current drug-regimen is complicated and of long-duration requiring a combination of the four rst-line drugs over a period of 6–9 months.[2] Recently, there has been an increase in the emergence of numerous drug-resistant strains which further complicates the regimen, prolongs treatment and is more expensive to administer. Extensively-drug resistant (XDRTB) strains of TB have been reported in 123 countries and there are o en no therapeutic agents to successfully treat these TB cases.[1] an increased knowledge of biochemical pathways employed by the Mtb pathogen is urgently required in order to develop new anti-tubercular agents with novel modes of action to reduce the global health threat from TB
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