Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase.

GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST). rGST was expressed in Escherichia coli JM109, using a pGEX4T-3 vector, and several anti-rGST monoclonal antibodies were generated using hybridoma technology. Two of these were rationally selected as capture and detection antibodies, allowing the development of a sandwich ELISA with a limit of detection (LOD) of 0.01 µg/ml. To develop the rGST-IPCR assay, we selected "Universal-IPCR" format, comprising the biotin-avidin binding as the coupling system. In addition, the rGST-IPCR was developed in standard PCR tubes, and the surface adsorption of antibodies on PCR tubes, the optimal neutravidin concentrations, the generation of a reporter DNA and the concentration effect were studied and determined. Under optimized assay conditions, the rGST-IPCR assay provided a 100-fold increase in the LOD as well as an expanded working range, in comparison with rGST-ELISA. The proposed method exhibited great potentiality for application in several fields in which measurement of very low levels of GST is necessary, and might provide a model for other IPCR assays.

Echinococcus granulosus: Evidence of a heterodimeric glutathione transferase built up by phylogenetically distant subunits.

In the cestode parasite Echinococcus granulosus, three phylogenetically distant cytosolic glutathione transferases (GSTs) (EgGST1, 2 and 3) were identified. Interestingly, the C-terminal domains of EgGST3 and EgGST2 but not EgGST1, exhibit all amino acids involved in Sigma-class GST dimerization. Here, we provide evidence indicating that EgGST2 and EgGST3 naturally form a heterodimeric structure (EgGST2-3), and also we report the enzymatic activity of the recombinant heterodimer. EgGST2-3 might display novel properties able to influence the infection establishment. This is the first report of a stable heterodimeric GST built up by phylogenetically distant subunits.

Purification of a recombinant glutathione transferase from the causative agent of hydatidosis, Echinococcus granulosus.

This practical class activity was designed to introduce students to recombinant protein expression and purification. The principal goal is to shed light on basic aspects concerning recombinant protein production, in particular protein expression, chromatography methods for protein purification, and enzyme activity as a tool to evaluate purity and conformation of the recombinant product. Herein, we describe the purification of a glutathione transferase from the human parasite Echinococcus granulosus (EgGST1), the causative agent of hydatidosis. EgGST1 is expressed fused to a histidine tag and is purified by immobilized metal affinity chromatography. Protein quantification based on direct (UV absorbance) and indirect (colorimetric) methods are used and discussed. A simple colorimetric assay is used to measure GST activity and special emphasis is put on how to use these measurements to follow protein purification yields, its enrichment and its correct folding along the purification process. EgGST1 is easily expressed with high yields, purified in absence of protease inhibitors and proved to be robust concerning enzyme activity and protein integrity on a 1 week practical activity.

Detection of anti-oxidant enzymatic activities and purification of glutathione transferases from Angiostrongylus cantonensis.

There are several anti-oxidant enzyme families that play pivotal roles in facilitating the survival of parasites. Glutathione transferases (GSTs) are members of the anti-oxidant family that can detoxify a broad range of exogenous or endogenous compounds including reactive oxidative species. GSTs have been studied as vaccine candidates, immunodiagnostic markers and as treatment targets. Helminths of the genus Angiostrongylus live inside arteries of vertebrates and two main species are associated with accidental human infections: Angiostrongylus costaricensis adult worms live inside the mesenteric arteries and larvae of Angiostrongylus cantonensis become trapped in the central nervous system vasculature. Since the interactions between angiostrongylid nematodes and their vertebrate hosts are poorly understood, this study characterized the anti-oxidant enzymatic activities of A. cantonensis from female worms by collecting excreted and secreted (ES) and total extract (TE) molecules. Catalase (CAT) and superoxide dismutase (SOD) activities were found both in the ES and TE while glutathione peroxidase (GPX) and GST were found only in the TE. GSTs were purified by glutathione agarose affinity column (AcGST) and the pool of eluted GSTs was analyzed by mass spectrometry (LC-MS/MS) and de novo sequencing (Masslynx software). Sequences from two peptides (AcGSTpep1 and AcGSTpep2) present high identity to the N-terminal and C-terminal from sigma class GSTs of nematodes. It is known that these GST enzymes are associated with host immune regulation. Furthermore, understanding the role of parasite-derived anti-oxidant molecules is important in understanding host-parasite interactions.

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa.

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST.

Characterization of Taenia solium cysticerci microsomal glutathione S-transferase activity.

Glutathione S-transferase activity has been shown to be associated with the microsomal fraction of Taenia solium. Electron microscopy and subcellular enzyme markers indicate the purity of the microsomal fraction that contains the glutathione S-transferase activity. T. solium microsomes were solubilized under conditions used to solubilize integral microsomal proteins. This procedure proved necessary to obtain enzymatic activity. To characterize this parasite enzyme activity, several substrates and inhibitors were used. The optimum activity for microsomal glutathione S-transferase was found to be pH 6.6, with a specific enzyme activity of 0.9, 0.1, 0.067, 0.03, and 0.05 micromol min(-1) mg(-1) with the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 4-hydroxynonenal, 2,4-hexadienal, and trans-2-nonenal, respectively. No activity of glutathione peroxidase was observed. T. solium microsomes had an appKm (GSH)=0.161 microM, appKm (CDNB)=14.5 microM, and appVmax of 0.15 and 27.9 micromol min(-1) mg(-1) for GSH and CDNB, respectively. T. solium microsomes were inhibited by several glutathione S-transferase enzyme inhibitors, and it was possible to establish a simple inhibition system as well as corresponding Ki's for each inhibitor. These results indicate that the T. solium microsomal glutathione S-transferase is different from the parasite cytoplasmic enzymes that catalyze similar reactions.

Genetic polymorphism of GSTM1 in women with breast cancer and interact with reproductive history and several clinical pathologies

Due to the conflicting results regarding the association between breast cancerand the GSTM1 null mutation, our aim was to research this associationin a Brazilian population and correlations withsmoking, reproductive history and several clinical pathologies. A case-control study was performed on 105 women with breast cancer and 278 controls. Extraction of DNA was accomplished according to the protocol of the GFX© kit and polymorphism analysis by the PCR technique. The control and experimental groups were compared and statistical analysis assessed by Xy or Fisher's exact test. The deletion in the GSTM1 gene in the breast cancer group had a prevalence of 32 (30.4 percent) individuals with the presence of null mutation. In the control group, the null mutation was present in 104 (37.4 percent) women. Upon comparison of the two groups, no statistically significant difference of the GSTM1 gene was observed, with an odds ratio (OR) of 0.74, 95 percent, confidence interval (CI) 0.45 - 1.20, p = 0.277. The results conclusively show that singlegene GSTM1 polymorphisms do not confer a substantial risk of breastcancer to its carriers. Furthermore, in this study no correlation was found between GSTs andsmoking, reproductive history and several clinical pathologies with respect to cancer risk.

Purification, characterization and kinetic properties of the Taenia solium glutathione S-transferase isoform 26.5 kDa.

Glutathione S-transferases are major phase II detoxification enzymes. Taenia solium, a parasite of humans and pigs, is exposed to toxic products. The aim of this work was to purify and characterize a T. solium glutathione S-transferase isoform of 26.5 kDa (SGST26.5) in order to obtain its kinetic parameters. Homogeneous SGST26.5 was obtained by a simple purification procedure. SGST26.5 showed a p I of 7.07, and a native Mr of 60 kDa with 26.5 kDa subunits. The optimum activity for SGST26.5 was found at pH 6.5-7.0 in the range 10-42 degrees C. SGST26.5 had a specific enzyme activity of 78, 7.1, 6.6, and 0.7 microM min(-1) mg(-1) with CDNB, 1,2-dichloro-4-nitrobenzene, 2,4-hexadienal and trans-2-nonenal as substrates, respectively. It also had a kcat/ K(mCDNB)=2.15 x 10(3) M(-1 )s(-1), kcat/ KmGSH)=4.5 x 10(3) M(-1 )s(-1) and Vmax for GSH and CDNB=74 and 77 microM min(-1) mg(-1), respectively. SGST26.5 was inhibited in a noncompetitive form by cibacron blue, bromosulfophthalein and triphenyltin chloride. Inhibition studies as a function of inhibitor concentration show that the enzyme is a homodimer. Bireactant system analysis show that it follows an ordered sequential mechanism.

Protein depletion and refeeding change the proportion of mouse liver glutathione S-transferase subunits.

The effect of protein depletion followed by refeeding with a normal diet on the content of mouse liver cytosolic proteins was studied. By peptide-mass fingerprinting and N-terminal sequencing, three polypeptides whose contents changed with dietary protein level were identified as glutathione S-transferases (GST) Yb1, Yc and Yf subunits. Five days of depletion caused the increase of Yb1 and Yf (21.6% and 78.5%, respectively) and the decrease of Yc (31.2%). After two days of refeeding, Yb1 and Yc were practically restored, while the neoplastic marker Yf remained higher (63.4%). None of the nutritional conditions tested induced new GSTs. While protein depletion-refeeding altered the ratios between the constitutive GST subunits, total liver GST content and activity were unaffected by depletion and slightly increased by refeeding. The increased amounts of Yb1 and Yf, and the maintenance of total GST content, indicate that during protein depletion, the GST subunits levels are controlled by mechanisms different from the majority of cytosolic proteins.

A procedure for the generation and the purification of Escherichia coli thioredoxins with variable N-terminal sequences.

We have developed a rapid and simple procedure for the production and the purification of Escherichia coli thioredoxins containing additional amino acid residues at the N-terminus. By the polymerase chain reaction, the complete gene encoding for E. coli thioredoxin was modified and amplified with the addition at its 5' end of a BamHI cloning site and a triplet coding for an arginine residue instead of the initiator methionine codon, whereas at the 3' end the stop codon was followed by an EcoRI cloning site. The synthetic DNA was ligated into the BamHI/EcoRI site of the vector plasmid pGEX-2T, and the novel plasmid [pFTG] was used for the transformation of E. coli cells. Following induction and cell disruption, a protein composed of Schistosoma japonicum glutathione S-transferase and E. coli thioredoxin was obtained in soluble form and purified by affinity chromatography on agarose columns bearing immobilized glutathione. This procedure yielded 50 mg of homogeneous fusion protein per liter of culture media. Digestion of the chimeric thioredoxin with bovine plasma thrombin followed by an additional chromatography on glutathione-agarose gave a protein that contained the entire sequence of E. coli thioredoxin and three additional amino acid residues [G-S-R-] at the N-terminal side. The structural characteristics and the protein disulfide oxidoreductase activity of this recombinant protein, in terms of variations of emission fluorescence and reduction of insulin disulfide bonds, respectively, were essentially identical to those of its counterpart obtained from wild-type cells by conventional techniques of proteins purification.(ABSTRACT TRUNCATED AT 250 WORDS)

One-step purification of plant ferredoxin-NADP+ oxidoreductase expressed in Escherichia coli as fusion with glutathione S-transferase.

Complementary DNA sequences encoding the mature form of pea ferredoxin-NADP+ reductase were cloned in-frame at the 3' end of the Schistosoma japonicum glutathione S-transferase gene in the expression vector pGEX-3X (Smith and Johnson, Gene 67, 31-40, 1988). A spacer sequence linking the two genes was modified to provide a proteolytic site just before the first amino acid residue of mature pea reductase. When introduced into competent Escherichia coli cells and induced, the resulting plasmid (pGF205) directed the expression of a 60-kDa immunoreactive peptide that results from the fusion between glutathione S-transferase and ferredoxin-NADP+ reductase sequences. The fused protein could be purified in a single step by selective absorption onto glutathione-agarose beads, followed by elution with free glutathione. It showed both transferase and reductase activities. Removal of the transferase portion by cleavage with the restriction protease Xa rendered ferredoxin-NADP+ reductase electrophoretically homogeneous. The purified transgenic enzyme showed kinetic and spectroscopic properties that were similar to those reported for the plant flavoprotein, indicating that, even when fused to the 27-kDa transferase portion, the reductase was still able to assemble FAD and to acquire an active conformation in the bacterial host. The expression-purification protocol employed here allows the isolation of up to 1 mg of active ferredoxin-NADP+ reductase/g of transformed cells. The system is potentially useful for the purification of activity-impaired forms of the flavoprotein.

Glutathione conjugation of microsome-mediated and synthetic aflatoxin B1-8,9-oxide by purified glutathione S-transferases from rats.

Glutathione (GSH) conjugation of microsome-mediated and synthetic aflatoxin B1 (AFB1)-epoxide and styrene oxide has been investigated with purified GSH S-transferases (GSTs) from rats. Both styrene oxide and AFB1-epoxide were conjugated preferentially by millimicrons GSTs 3-3, 3-4 and 4-4 as compared to alpha GSTs 1-1, 1-2 and 2-2. The highest catalytic activity with styrene oxide conjugation was associated with GST 4-4. The highest catalytic activity with microsome-mediated AFB1-epoxide conjugation was observed with GST 3-3 whereas with the synthetic AFB1-epoxide conjugation was seen with GST 4-4. The catalytic activity of pi GST 7-7 was intermediate to millimicrons and alpha GSTs. It is suggested that GST 3-3 may play an important role in inactivation of AFB1-epoxide generated in vivo in the rat.