title: Cloning, purification, crystallization and preliminary X-ray analysis of ESX-1-secreted protein regulator (EspR) from Mycobacterium tuberculosis.
abstract: 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 P3(1)21 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.
authors.affiliation: Rm-403/440, Structural Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India.
authors.forename: Shanti P
authors.initials: SP
authors.lastname: Gangwar
authors.forename: Sita R
authors.initials: SR
authors.lastname: Meena
authors.forename: Ajay K
authors.initials: AK
authors.lastname: Saxena
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D003460:Crystallization; D018398:Homeodomain Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry*; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D018398:Homeodomain Proteins