SapM from Mycobacterium tuberculosis is a secreted phosphatase critical for pathogen survival inside the host, representing an attractive target for the development of anti-tuberculosis drugs. The main limitation to biochemical and structural studies of SapM has been the lack of a suitable protocol to produce soluble recombinant protein. The aim of the present work was to produce SapM in Escherichia coli in a soluble and catalytically active form. We describe here the construct design, expression and purification of soluble SapM using Sarkosyl as a solubility-enhancing agent and auto-induction media. We demonstrate that solubilisation of the recombinant protein with Sarkosyl, and further purification, yields a catalytically active enzyme with high purity and monodisperse. The identity and molecular weight of the recombinant SapM was confirmed by mass spectrometry analyses, and we provide evidence that SapM behaves as a monomer in solution. Overall, this work lays the foundation for further studies to exploit SapM as a drug target, and provides a protocol for producing active and soluble recombinant enzymes that are hard to solubilise in E. coli.
Keywords: Active enzyme; Protein expression; Protein purification; SapM; Sarkosyl; Soluble protein; Tuberculosis.
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