Cloning, purification, crystallization and preliminary X-ray analysis of ESX-1-secreted protein regulator (EspR) from Mycobacterium tuberculosis

Shanti Gangwar, Sita R. Meena, Ajay K. Saxena

Research output: Contribution to journalArticle

2 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)83-86
Number of pages4
JournalActa Crystallographica Section F: Structural Biology and Crystallization Communications
Volume67
Issue number1
DOIs
StatePublished - Jan 2011
Externally publishedYes

Fingerprint

tuberculosis
Cloning
regulators
X ray analysis
Crystallization
Mycobacterium tuberculosis
purification
Purification
Organism Cloning
X-Rays
crystallization
proteins
Proteins
x rays
chromatography
Crystals
Synchrotrons
Affinity chromatography
Bacterial Proteins
Escherichia coli Proteins

Keywords

  • ESX-1-secreted protein regulator
  • Mycobacterium tuberculosis
  • Rv3849

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Genetics
  • Structural Biology
  • Condensed Matter Physics

Cite this

@article{5505383fff4f4c5b8b4d52ed743b2431,
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 {\AA} resolution using synchrotron radiation of wavelength 0.97625 {\AA}. 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.",
keywords = "ESX-1-secreted protein regulator, Mycobacterium tuberculosis, Rv3849",
author = "Shanti Gangwar and Meena, {Sita R.} and Saxena, {Ajay K.}",
year = "2011",
month = "1",
doi = "10.1107/S174430911004618X",
language = "English (US)",
volume = "67",
pages = "83--86",
journal = "Acta Crystallographica Section F:Structural Biology Communications",
issn = "1744-3091",
publisher = "John Wiley and Sons Ltd",
number = "1",

}

TY - JOUR

T1 - Cloning, purification, crystallization and preliminary X-ray analysis of ESX-1-secreted protein regulator (EspR) from Mycobacterium tuberculosis

AU - Gangwar, Shanti

AU - Meena, Sita R.

AU - Saxena, Ajay K.

PY - 2011/1

Y1 - 2011/1

N2 - 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.

AB - 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.

KW - ESX-1-secreted protein regulator

KW - Mycobacterium tuberculosis

KW - Rv3849

UR - http://www.scopus.com/inward/record.url?scp=78651103361&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=78651103361&partnerID=8YFLogxK

U2 - 10.1107/S174430911004618X

DO - 10.1107/S174430911004618X

M3 - Article

VL - 67

SP - 83

EP - 86

JO - Acta Crystallographica Section F:Structural Biology Communications

JF - Acta Crystallographica Section F:Structural Biology Communications

SN - 1744-3091

IS - 1

ER -