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Expression, purification, crystallization and structure determination of two glutathione S-transferase-like proteins from Shewanella oneidensis.

ABSTRACT: Genome analysis of Shewanella oneidensis, a Gram-negative bacterium with an unusual repertoire of respiratory and redox capabilities, revealed the presence of six glutathione S-transferase-like genes (sogst1-sogst6). Glutathione S-transferases (GSTs; EC 2.5.1.18) are found in all kingdoms of life and are involved in phase II detoxification processes by catalyzing the nucleophilic attack of reduced glutathione on diverse electrophilic substrates, thereby decreasing their reactivity. Structure-function studies of prokaryotic GST-like proteins are surprisingly underrepresented in the scientific literature when compared with eukaryotic GSTs. Here, the production and purification of recombinant SoGST3 (SO_1576) and SoGST6 (SO_4697), two of the six GST-like proteins in S. oneidensis, are reported and preliminary crystallographic studies of crystals of the recombinant enzymes are presented. SoGST3 was crystallized in two different crystal forms in the presence of GSH and DTT that diffracted to high resolution: a primitive trigonal form in space group P3(1) that exhibited merohedral twinning with a high twin fraction and a primitive monoclinic form in space group P2(1). SoGST6 yielded primitive orthorhombic crystals in space group P2(1)2(1)2(1) from which diffraction data could be collected to medium resolution after application of cryo-annealing protocols. Crystal structures of both SoGST3 and SoGST6 have been determined based on marginal search models by maximum-likelihood molecular replacement as implemented in the program Phaser.

SUBMITTER: Remmerie B

PROVIDER: S-EPMC2496851 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Expression, purification, crystallization and structure determination of two glutathione S-transferase-like proteins from Shewanella oneidensis.

Remmerie Bert B Vandenbroucke Karen K De Smet Lina L Carpentier Wesley W De Vos Dirk D Stout Jan J Van Beeumen Jozef J Savvides Savvas N SN

Acta crystallographica. Section F, Structural biology and crystallization communications 20080524 Pt 6

Genome analysis of Shewanella oneidensis, a Gram-negative bacterium with an unusual repertoire of respiratory and redox capabilities, revealed the presence of six glutathione S-transferase-like genes (sogst1-sogst6). Glutathione S-transferases (GSTs; EC 2.5.1.18) are found in all kingdoms of life and are involved in phase II detoxification processes by catalyzing the nucleophilic attack of reduced glutathione on diverse electrophilic substrates, thereby decreasing their reactivity. Structure-fun ...[more]

PMID: 18540073

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Project description:A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 microg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 microg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 microg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10-50 ml) cell growth and automated purification. 1H-15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA-GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 A. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.

Dataset Information

Expression, purification, crystallization and structure determination of two glutathione S-transferase-like proteins from Shewanella oneidensis.

Publications

Expression, purification, crystallization and structure determination of two glutathione S-transferase-like proteins from Shewanella oneidensis.

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