Cloning, purification, crystallization and preliminary X‐ray analysis of ESX‐1‐secreted protein regulator (EspR) from Mycobacterium tuberculosis
ESX‐1‐secreted protein regulator (EspR; Rv3849) is a key regulator in Mycobacterium tuberculosis that delivers bacterial proteins into the host cell during infection. EspR binds directly to the Rv3616c‐Rv3614c promoter and activates transcription and secretes itself from the bacterial cell by the ESX‐1 system. The three‐dimensional structure of EspR will aid in understanding the mechanisms by which it binds to the Rv3616c‐Rv3614c promoter and is involved in transcriptional activation. This study will significantly aid in the development of EspR‐based therapeutics against M. tuberculosis. The full‐length EspR gene from M. tuberculosis (H37Rv strain) was cloned and overexpressed as a soluble protein in Escherichia coli. The protein was purified by affinity chromatography using His‐tagged protein followed by size‐exclusion chromatography. EspR was crystallized using polyethylene glycol 3350 as precipitant. The crystals diffracted to 3.2 Å resolution using synchrotron radiation of wavelength 0.97625 Å. The crystal belonged to space group P3121 and contained three monomers in the asymmetric unit. Native and heavy‐atom‐derivatized data sets were collected from EspR crystals for use in ab initio structure‐solution techniques.
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