skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Cloning, expression, purification, crystallization and initial crystallographic analysis of transcription elongation factors GreB from Escherichia coli and Gfh1 from Thermus thermophilus

Abstract

The GreB transcription cleavage factor of E. coli and the Gfh1 transcription elongation factor of T. thermophilus were cloned, expressed, purified and crystallized. Complete diffraction data sets were collected for the GreB and Gfh1 crystals to 2.6 and 2.8 Å resolution, respectively. Structure determination of these proteins is now in progress.

Authors:
; ; ; ; ;
Publication Date:
OSTI Identifier:
22355954
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 62; Journal Issue: Pt 1; Other Information: PMCID: PMC1401493; PMID: 16511259; PUBLISHER-ID: pu5114; OAI: oai:pubmedcentral.nih.gov:1401493; Copyright (c) International Union of Crystallography 2006; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CLEAVAGE; CRYSTALLIZATION; CRYSTALS; DIFFRACTION; ELONGATION; ESCHERICHIA COLI; PROTEINS; RESOLUTION

Citation Formats

Perederina, Anna A., Vassylyeva, Marina N., Berezin, Igor A., Svetlov, Vladimir, Artsimovitch, Irina, and Vassylyev, Dmitry G., E-mail: dmitry@uab.edu. Cloning, expression, purification, crystallization and initial crystallographic analysis of transcription elongation factors GreB from Escherichia coli and Gfh1 from Thermus thermophilus. United Kingdom: N. p., 2006. Web. doi:10.1107/S1744309105040297.
Perederina, Anna A., Vassylyeva, Marina N., Berezin, Igor A., Svetlov, Vladimir, Artsimovitch, Irina, & Vassylyev, Dmitry G., E-mail: dmitry@uab.edu. Cloning, expression, purification, crystallization and initial crystallographic analysis of transcription elongation factors GreB from Escherichia coli and Gfh1 from Thermus thermophilus. United Kingdom. doi:10.1107/S1744309105040297.
Perederina, Anna A., Vassylyeva, Marina N., Berezin, Igor A., Svetlov, Vladimir, Artsimovitch, Irina, and Vassylyev, Dmitry G., E-mail: dmitry@uab.edu. Sun . "Cloning, expression, purification, crystallization and initial crystallographic analysis of transcription elongation factors GreB from Escherichia coli and Gfh1 from Thermus thermophilus". United Kingdom. doi:10.1107/S1744309105040297.
@article{osti_22355954,
title = {Cloning, expression, purification, crystallization and initial crystallographic analysis of transcription elongation factors GreB from Escherichia coli and Gfh1 from Thermus thermophilus},
author = {Perederina, Anna A. and Vassylyeva, Marina N. and Berezin, Igor A. and Svetlov, Vladimir and Artsimovitch, Irina and Vassylyev, Dmitry G., E-mail: dmitry@uab.edu},
abstractNote = {The GreB transcription cleavage factor of E. coli and the Gfh1 transcription elongation factor of T. thermophilus were cloned, expressed, purified and crystallized. Complete diffraction data sets were collected for the GreB and Gfh1 crystals to 2.6 and 2.8 Å resolution, respectively. Structure determination of these proteins is now in progress.},
doi = {10.1107/S1744309105040297},
journal = {Acta Crystallographica. Section F},
number = Pt 1,
volume = 62,
place = {United Kingdom},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
  • The SecA ATPase from T. thermophilus was cloned, expressed, purified and crystallized. Complete diffraction data sets were collected for two crystal forms at 2.8 and 3.5 Å resolution, respectively. Determination of the structure is now in progress. The Thermus thermophilus gene encoding the preprotein translocation ATPase SecA was cloned and expressed and the purified protein was crystallized by the hanging-drop vapour-diffusion technique in two different space groups P3{sub 1(2)}21 (a = b = 168.6, c = 149.8 Å) and P6{sub 1(5)}22 (a = b = 130.9, c = 564.6 Å). The crystals, improved by macroseeding, diffracted to beyond 2.8 andmore » 3.5 Å resolution for the trigonal and hexagonal crystal forms, respectively. Structure determination using the multiple isomorphous replacement method is in progress.« less
  • The molybdopterin synthase from T. thermophilus HB8 was cloned, expressed, purified and crystallized. The crystals belong to space group P2{sub 1} and diffracted to a resolution of 1.64 Å. Thermus thermophilus is a Gram-negative aerobic thermophilic eubacterium which can grow at temperatures ranging from 323 to 355 K. In addition to their importance in thermostability or adaptation strategies for survival at high temperatures, the thermostable enzymes in thermophilic organisms contribute to a wide range of biotechnological applications. The molybdenum cofactor in all three kingdoms consists of a tricyclic pyranopterin termed molybdopterin that bears the cis-dithiolene group responsible for molybdenum ligation.more » The crystals of molybdopterin synthase from T. thermophilus HB8 belong to the primitive monoclinic space group P2{sub 1}, with unit-cell parameters a = 33.94, b = 103.32, c = 59.59 Å, β = 101.3°. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit.« less
  • Acetylornithine aminotransferases, members of the type I subgroup II family of PLP-dependent enzymes, from S. typhimurium and E. coli have been cloned, overexpressed, purified and crystallized. Acetylornithine aminotransferase (AcOAT) is a type I pyridoxal 5′-phosphate-dependent enzyme catalyzing the conversion of N-acetylglutamic semialdehyde to N-acetylornithine in the presence of α-ketoglutarate, a step involved in arginine metabolism. In Escherichia coli, the biosynthetic AcOAT also catalyzes the conversion of N-succinyl-l-2-amino-6-oxopimelate to N-succinyl-l,l-diaminopimelate, one of the steps in lysine biosynthesis. It is closely related to ornithine aminotransferase. AcOAT was cloned from Salmonella typhimurium and E. coli, overexpressed in E. coli and purified using Ni–NTAmore » affinity column chromatography. The enzymes crystallized in the presence of gabaculine. Crystals of E. coli AcOAT (eAcOAT) only diffracted X-rays to 3.5 Å and were twinned. The crystals of S. typhimurium AcOAT (sAcOAT) diffracted to 1.9 Å and had a dimer in the asymmetric unit. The structure of sAcOAT was solved by the molecular-replacement method.« less
  • The transcriptional regulator AcrR from Escherichia coli has been cloned, overexpressed, purified and crystallized and X-ray diffraction data have been collected to a resolution of 2.5 Å. This paper describes the cloning, expression, purification and preliminary X-ray data analysis of the AcrR regulatory protein. The Escherichia coli AcrR is a member of the TetR family of transcriptional regulators. It regulates the expression of the AcrAB multidrug transporter. Recombinant AcrR with a 6×His tag at the C-terminus was expressed in E. coli and purified by metal-affinity chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected frommore » cryocooled crystals at a synchrotron light source. The best crystal diffracted to 2.5 Å. The space group was determined to be P3{sub 2}, with unit-cell parameters a = b = 46.61, c = 166.16 Å.« less
  • The crystallization and preliminary X-ray diffraction analysis of the l-fuculose-1-phosphate aldolase (FucA) from T. thermophilus HB8. Native diffraction data set was collected to a resolution of 1.9 Å. Fuculose phosphate aldolase catalyzes the reversible cleavage of l-fuculose-1-phosphate to dihydroxyacetone phosphate and l-lactaldehyde. The protein from Thermus thermophilus HB8 is a biological tetramer with a subunit molecular weight of 21 591 Da. Purified FucA has been crystallized using sitting-drop vapour-diffusion and microbatch techniques at 293 K. The crystals belong to space group P4, with unit-cell parameters a = b = 100.94, c = 45.87 Å. The presence of a dimer ofmore » the enzyme in the asymmetric unit was estimated to give a Matthews coefficient (V{sub M}) of 2.7 Å{sup 3} Da{sup −1} and a solvent content of 54.2%(v/v). Three-wavelength diffraction MAD data were collected to 2.3 Å from zinc-containing crystals. Native diffraction data to 1.9 Å resolution have been collected using synchrotron radiation at SPring-8.« less