Functional Genomics, the systematic characterisation of the functions of an organism's genes, includes the study of the gene products, the proteins. Such studies require methods to express and purify these proteins in a parallel, time and cost effective manner.
We developed a method for parallel expression and purification of recombinant proteins with a hexahistidine tag (His-tag) or glutathione S-transferase (GST)-tag from bacterial expression systems. Proteins are expressed in 96-well microplates and are purified by a fully automated procedure on a pipetting robot. Up to 90 microgram purified protein can be obtained from 1 ml microplate cultures. The procedure is readily reproducible and 96 proteins can be purified in approximately three hours. It avoids clearing of crude cellular lysates and the use of magnetic affinity beads and is therefore less expensive than comparable commercial systems.
We have used this method to compare purification of a set of human proteins via His-tag or GST-tag. Proteins were expressed as fusions to an N-terminal tandem His- and GST-tag and were purified by metal chelating or glutathione affinity chromatography. The purity of the obtained protein samples was similar, yet His-tag purification resulted in higher yields for some proteins.
A fully automated, robust and cost effective method was developed for the purification of proteins that can be used to quickly characterise expression clones in high throughput and to produce large numbers of proteins for functional studies.
His-tag affinity purification was found to be more efficient than purification via GST-tag for some proteins.
Hydrogels are finding increased clinical utility as advances continue to exploit their favorable material properties. Hydrogels can be adapted for many applications, including surface coatings and drug delivery. Anti-infectious surfaces and delivery systems that actively destroy invading organisms are alternative ways to exploit the favorable material properties offered by hydrogels. Sterilization techniques are commonly employed to ensure the materials are non-infectious upon placement, but sterilization is not absolute and infections are still expected. Natural, anti-bacterial proteins have been discovered which have the potential to act as anti-infectious agents; however, the proteins are toxic and need localized release to have therapeutic efficacy without toxicity. In these studies, we explore the use of the glutathione s-transferase (GST) to anchor the bactericidal peptide, melittin, to the surface of poly(ethylene glycol) diacrylate (PEGDA) hydrogel microspheres. We show that therapeutic levels of protein can be anchored to the surface of the microspheres using the GST anchor. We compared the therapeutic efficacy of recombinant melittin released from PEGDA microspheres to melittin. We found that, when released by an activating enzyme, thrombin, recombinant melittin efficiently inhibits growth of the pathogenic bacterium Streptococcus pyogenes as effectively as melittin created by solid phase peptide synthesis. We conclude that a GST protein anchor can be used to immobilize functional protein to PEGDA microspheres and the protein will remain immobilized under physiological conditions until the protein is enzymatically released.
recombinant protein; glutathione s-transferase; glutathione; thrombin; hydrogel; microparticles
Amyloid fibrils of Alzheimer’s β-amyloid peptide (Aβ) are a primary component of amyloid plaques, a hallmark of Alzheimer’s disease (AD). Enormous attention has been given to the structural features and functions of Aβ in amyloid fibrils and other type of aggregates in associated with development of AD. This report describes an efficient protocol to express and purify high-quality 40-residue Aβ(1–40), the most abundant Aβ in brains, for structural studies by NMR spectroscopy. Over-expression of Aβ(1–40) with glutathione S-transferase (GST) tag connected by a Factor Xa recognition site (IEGR▼) in E. Coli resulted in the formation of insoluble inclusion bodies even with the soluble GST tag. This problem was resolved by efficient recovery of the GST-Aβ fusion protein from the inclusion bodies using 0.5% (w/v) sodium lauroyl sarcosinate as solubilizing agent and subsequent purification by affinity chromatography using a glutathione agarose column. The removal of the GST tag by Factor Xa enzymatic cleavage and purification by HPLC yielded as much as ~7 mg and ~1.5 mg of unlabeled Aβ(1–40) and uniformly 15N- and/or 13C-protein Aβ(1–40) from 1 L of the cell culture, respectively. Mass spectroscopy of unlabeled and labeled Aβ and 1H/15N HSQC solution NMR spectrum of the obtained 15N-labeled Aβ in the monomeric form confirmed the expression of native Aβ(1–40). It was also confirmed by electron micrography and solid-state NMR analysis that the purified Aβ(1–40) self-assembles into β-sheet rich amyloid fibrils. To the best of our knowledge, our protocol offers the highest yields among published protocols for production of recombinant Aβ(1–40) samples that are amendable for an NMR-based structural analysis. The protocol may be applied to efficient preparation of other amyloid-forming proteins and peptides that are 13C- and 15N-labeled for NMR experiments.
Amyloid β; GST fusion protein; sodium lauroyl sarcosinate; NMR
Drosophila ribosomal protein PO was overexpressed in Escherichia coli to allow for its purification, biochemical characterization and to generate polyclonal antibodies for Western analysis. Biochemical tests were originally performed to see if overexpressed PO contained DNase activity similar to that recently reported for the apurinic/apyrimidinic (AP) lyase activity associated with Drosophila ribosomal protein S3. The overexpressed ribosomal protein was subsequently found to act on AP DNA, producing scissions that were in this case 5' of a baseless site instead of 3', as has been observed for S3. As a means of confirming that the source of AP endonuclease activity was in fact due to PO, glutathione S-transferase (GST) fusions containing a Factor Xa cleavage site between GST and PO were constructed, overexpressed in an E.coli strain defective for the major 5'-acting AP endonucleases and the fusions purified using glutathione-agarose affinity column chromatography. Isolated fractions containing purified GST-PO fusion proteins were subsequently found to have authentic AP endonuclease activity. Moreover, glutathione-agarose was able to deplete AP endonuclease activity from GST-PO fusion protein preparations, whereas the resin was ineffective in lowering DNA repair activity for PO that had been liberated from the fusion construct by Factor Xa cleavage. These results suggested that PO was a multifunctional protein with possible roles in DNA repair beyond its known participation in protein translation. In support of this notion, tests were performed that show that GST-PO, but not GST, was able to rescue an E.coli mutant lacking the major 5'-acting AP endonucleases from sensitivity to an alkylating agent. We furthermore show that GST-PO can be located in both the nucleus and ribosomes. Its nuclear location can be further traced to the nuclear matrix, thus placing PO in a subcellular location where it could act as a DNA repair protein. Other roles beyond DNA repair seem possible, however, since GST-PO also exhibited significant nuclease activity for both single- and double-stranded DNA.
This chapter describes the use of glutathione S-transferase (GST) gene fusion proteins as a method for inducible, high-level protein expression and purification from bacterial cell lysates. The protein is expressed in a pGEX vector, with the GST moiety located at the N-terminus followed by the target protein. The use of GST as a fusion tag is desirable because it can act as a chaperone to facilitate protein folding, and frequently the fusion protein can be expressed as a soluble protein rather than in inclusion bodies. Additionally, the GST fusion protein can be affinity purified facilely without denaturation or use of mild detergents. The fusion protein is captured by immobilized glutathione and impurities are washed away. The fusion protein then is eluted under mild, non-denaturing conditions using reduced glutathione. If desired, the removal of the GST affinity tag is accomplished by using a site-specific protease recognition sequence located between the GST moiety and the target protein. Purified proteins have been used successfully in immunological studies, structure determinations, vaccine production, protein-protein, and protein-DNA interaction studies and other biochemical analysis.
Glutathione S-transferase (GST); pGEX; protein expression; protein purification; thrombin; factor Xa; fusion tags
The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases. Escherichia coli DH5α and Vibrio harveyi KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined. A fusion between glutathione S-transferase (GST) and E. coli DH5α NfsA (GST-EcNfsA), a fusion between GST and E. coli DH5α NfsB (GST-EcNfsB), and a fusion between GST and V. harveyi KCTC 2720 NfsA (GST-VhNfsA) were prepared for their overproduction and easy purification. GST-EcNfsA, GST-EcNFsB, and GST-VhNFsA efficiently reduced nitrofurazone and 2,4,6-trinitrotoluene (TNT) as their nitro substrates. The Km values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 11.8, 23.5, and 5.4 μM, respectively. The Vmax values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were 3.8, 3.9, and 10.7 nmol/min/mg of protein, respectively. GST-VhNfsA was the most effective of the three chromate reductases, as determined by each Vmax/Km value. The optimal temperatures of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 55, 30, and 30°C, respectively. Thus, it is confirmed that nitroreductase can also act as a chromate reductase. Nitroreductases may be used in chromate remediation. GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA have a molecular mass of 50 kDa and exist as a monomer in solution. Thin-layer chromatography showed that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA contain FMN as a cofactor. GST-VhNfsA reduced Cr(VI) to Cr(III). Cr(III) was much less toxic to E. coli than Cr(VI).
Silkworm β-1,3-glucan recognition protein (βGRP) tightly and specifically associates with β-1,3-glucan. We report here an affinity purification system named the ‘GRP system’, which uses the association between the β-1,3-glucan recognition domain of βGRP (GRP-tag), as an affinity tag, and curdlan beads. Curdlan is a water-insoluble β-1,3-glucan reagent, the low cost of which (about 100 JPY/g) allows the economical preparation of beads. Curdlan beads can be readily prepared by solubilization in an alkaline solution, followed by neutralization, sonication and centrifugation. We applied the GRP system to preparation of several proteins and revealed that the expression levels of the GRP-tagged proteins in soluble fractions were two or three times higher than those of the glutathione S-transferase (GST)-tagged proteins. The purity of the GRP-tagged proteins on the curdlan beads was comparable to that of the GST-tagged proteins on glutathione beads. The chemical stability of the GRP system was more robust than conventional affinity systems under various conditions, including low pH (4–6). Biochemical and structural analyses revealed that proteins produced using the GRP system were structurally and functionally active. Thus, the GRP system is suitable for both the large- and small-scale preparation of recombinant proteins for functional and structural analyses.
β-1,3-glucan recognition protein; affinity tag; curdlan; glutathione S-transferase; recombinant protein
site-selective conjugation of poly(ethylene glycol)
(PEG) to the glutathione (GSH) binding pocket of glutathione S-transferase (GST) is described. To achieve this, a GSH
analogue (GSH-BP) was designed and chemically synthesized with three
functionalities: (1) the binding affinity of GSH to GST, (2) a free
thiol for polymer functionalization, and (3) a photoreactive benzophenone
(BP) component. Different molecular weights (2 kDa, 5 kDa, and 20
kDa) of GSH-BP modified PEGs (GSBP-PEGs) were synthesized and showed
conjugation efficiencies between 52% and 76% to GST. Diazirine (DA)
PEG were also prepared but gave conjugation yields lower than for
GSBP-PEGs. PEGs with different end-groups were also synthesized to
validate the importance of each component in the end-group design.
End-groups included glutathione (GS-PEG) and benzophenone (BP-PEG).
Results showed that both GSH and BP were crucial for successful conjugation
to GST. In addition, conjugations of 5 kDa GSBP-PEG to different proteins
were investigated, including bovine serum albumin (BSA), lysozyme
(Lyz), ubiquitin (Ubq), and GST-fused ubiquitin (GST-Ubq) to ensure
specific binding to GST. By combining noncovalent and covalent interactions,
we have developed a new phototriggered protein–polymer conjugation
method that is generally applicable to GST-fusion proteins.
Era is an essential membrane-associated GTPase that is present in bacteria and mycoplasmas. Era appears to play an important role in the regulation of the bacterial cell cycle. In this study, we expressed the native and glutathione S-transferase (GST) fusion forms of Streptococcus pneumoniae Era in Escherichia coli and purified both proteins to homogeneity. We showed that RNA was copurified with the GST-Era protein of S. pneumoniae during affinity purification and remained associated with the protein after removal of the GST tag by thrombin cleavage. The thrombin-treated and untreated GST-Era proteins could bind and hydrolyze GTP and exhibited similar kinetic properties (dissociation constant [kD], Km, and Vmax). However, the native Era protein purified by using different chromatographic columns had a much lower GTPase activity than did GST-Era, although it had a similar kD. In addition, RNA was not associated with the protein. Purified GST-Era protein was shown to be present as high (600-kDa)- and low (120-kDa)-molecular-mass forms. The high-molecular-mass form of GST-Era was associated with RNA and exhibited a very high GTPase activity. Approximately 40% of purified GST-Era protein was associated with RNA, and removal of the RNA resulted in a significant reduction in GTPase activity. The RNA associated with GST-Era was shown to be predominantly 16S rRNA. The native Era protein isolated directly from S. pneumoniae was also present as a high-molecular-mass species (600 kDa) complexed with RNA. Together, our results suggest that 16S rRNA is associated with Era and might stimulate its GTPase activity.
The production and purification of recombinant SoGST3 and SoGST6, two GST-like proteins from S. oneidensis, are reported and preliminary crystallographic studies of crystals of the recombinant enzymes are presented.
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 220.127.116.11) 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 P31 that exhibited merohedral twinning with a high twin fraction and a primitive monoclinic form in space group P21. SoGST6 yielded primitive orthorhombic crystals in space group P212121 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.
glutathione S-transferases; Shewanella oneidensis
Genetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD). Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intestinal flora, it has been difficult to design experiments for detection of proinflammatory bacterial antigen(s) involved in the pathogenesis of the disease. Several studies indicated a potential association of E. coli with IBD. In addition, T cell clones of IBD patients were shown to cross react towards antigens from different enteric bacterial species and thus likely responded to conserved bacterial antigens. We therefore chose highly conserved E. coli proteins as candidate antigens for abnormal T cell responses in IBD and used high-throughput techniques for cloning, expression and purification under native conditions of a set of 271 conserved E. coli proteins for downstream immunologic studies.
As a standardized procedure, genes were PCR amplified and cloned into the expression vector pQTEV2 in order to express proteins N-terminally fused to a seven-histidine-tag. Initial small-scale expression and purification under native conditions by metal chelate affinity chromatography indicated that the vast majority of target proteins were purified in high yields. Targets that revealed low yields after purification probably due to weak solubility were shuttled into Gateway (Invitrogen) destination vectors in order to enhance solubility by N-terminal fusion of maltose binding protein (MBP), N-utilizing substance A (NusA), or glutathione S-transferase (GST) to the target protein. In addition, recombinant proteins were treated with polymyxin B coated magnetic beads in order to remove lipopolysaccharide (LPS). Thus, 73% of the targeted proteins could be expressed and purified in large-scale to give soluble proteins in the range of 500 μg.
Here, we report a cost-efficient procedure to produce around 200 soluble recombinant E. coli proteins in large-scale, including removal of LPS by polymyxin B coated beads for subsequent use of the proteins in downstream immunological studies.
AIM: To survey glutathione (GSH) S-transferase (GST) isoforms in mitochondria and to reveal the isoforms’ biological significance in diabetic mice.
METHODS: The presence of GSTs in mouse liver mitochondria was systematically screened by two proteomic approaches, namely, GSH affinity chromatography/two dimensional electrophoresis (2DE/MALDI TOF/TOF MS) and SDS-PAGE/LC ESI MS/MS. The proteomic results were further confirmed by Western blotting using monoclonal antibodies against GSTs. To evaluate the liver mitochondrial GSTs quantitatively, calibration curves were generated by the loading amounts of individual recombinant GST protein vs the relative intensities elicited from the Western blotting. An extensive comparison of the liver mitochondrial GSTs was conducted between normal and db/db diabetic mice. Student’s t test was adopted for the estimation of regression and significant difference.
RESULTS: Using GSH affinity/2DE/MALDI TOF/TOF MS, three GSTs, namely, alpha3, mu1 and pi1, were identified; whereas five GSTs, alpha3, mu1, pi1, kappa1 and zeta1, were detected in mouse liver mitochondria using SDS-PAGE/LC ESI MS/MS, of these GSTs, GST kappa1 was reported as a specific mitochondrial GST. The R2 values of regression ranged between values of about 0.86 and 0.98, which were acceptable for the quantification. Based on the measurement of the GST abundances in liver mitochondria of normal and diabetic mice, the four GSTs, alpha3, kappa1, mu1 and zeta1, were found to be almost comparable between the two sets of animals, whereas, lower GST pi1 was detected in the diabetic mice compared with normal ones, the signal of Western blotting in control and db/db diabetic mice liver mitochondria is 134.61 ± 53.84 vs 99.74 ± 46.2, with P < 0.05.
CONCLUSION: Our results indicate that GSTs exist widely in mitochondria and its abundances of mitochondrial GSTs might be tissue-dependent and disease-related.
Glutathione S-transferase; Mitochondria; Liver; Proteomics; Diabetes
Pigeon circovirus (PiCV) is considered to be a viral agent central to the development of young pigeon disease syndrome (YPDS). The Cap protein, a structural protein encoded by the cap (or C1) gene of PiCV, has been shown to be responsible for not only capsid assembly, but also has been used as antigen for detecting antibody when the host is infected with PiCV. The antigenic characteristics of the Cap protein potentially may allow the development of a detection kit that could be applied to control PiCV infection. However, poor expression and poor protein solubility have hampered the production of recombinant Cap protein in the bacteria. This study was undertaken to develop the optimal expression of recombinant full-length Cap protein of PiCV using an E. coli expression system.
The PiCV cap gene was cloned and fused with different fusion partners including a His-tag, a GST-tag (glutathioine-S-transferase tag) and a Trx-His-tag (thioredoxin-His tag). The resulting constructs were then expressed after transformation into a number of different E. coli strains; these then had their protein expression evaluated. The expression of the recombinant Cap protein in E. coli was significantly increased when Cap protein was fused with either a GST-tag or a Trx-His tag rather than a His-tag. After various rare amino acid codons presented in the Cap protein were optimized to give the sequence rCapopt, the expression level of the GST-rCapopt in E. coli BL21(DE3) was further increased to a significant degree. The highest protein expression level of GST-rCapopt obtained was 394.27 ± 26.1 mg/L per liter using the E. coli strain BL21(DE3)-pLysS. Moreover, approximately 74.5% of the expressed GST-rCapopt was in soluble form, which is higher than the soluble Trx-His-rCapopt expressed using the BL21(DE3)-pLysS strain. After purification using a GST affinity column combined with ion-exchange chromatography, the purified recombinant GST-rCapopt protein was found to have good antigenic activity when tested against PiCV-infected pigeon sera.
These findings shows that the E. coli-expressed full-length PiCV Cap protein has great potential in terms of large-scaled production and this should allow in the future the development of a serodiagnostic kit that is able to clinically detect PiCV infection in pigeons.
To study the interaction between the lens-specific water channel protein, aquaporin 0 (AQP0) and the lens-specific intermediate filament protein, filensin, and the effect of this interaction on the water permeability of AQP0. The effect of other factors on the interaction was also investigated.
Expression plasmids were constructed in which glutathione-S-transferase (GST) was fused to the AQP0 COOH-terminal region (GST-AQP0-C), which contains the major phosphorylation sites of the protein. Plasmids for AQP0 COOH-terminal mutants were also constructed in which one, three or five sites were pseudophosphorylated, and the proteins expressed from these GST-fusion plasmids were assayed for their interaction with lens proteins. Expressed recombinant GST-fusion proteins were purified using glutathione beads and incubated with rat lens extract. Western blotting was used to identify the lens proteins that interacted with the GST-fusion proteins. Filensin tail and rod domains were also expressed as GST-fusion proteins and their interactions with AQPO were analyzed. Additionally, the water permeability of AQP0 was calculated by expressing AQP0 with or without the filensin peptide on the cell membrane of Xenopus oocytes by injecting cRNAs for AQP0 and filensin.
The GST-AQP0-C construct interacted with the tail region of lens filensin and the GST-filensin-tail construct interacted with lens AQP0, but the GST-filensin-rod construct did not interact with AQP0. GST-AQP0-C also interacted with a purified recombinant filensin-tail peptide after cleavage from GST. The AQP0/filensin-tail interaction was not affected by pseudophosphorylation of the AQP0 COOH-terminal tail, nor was it affected by changes in pH. Xenopus oocytes expressing AQP0 on the plasma membrane showed increased water permeability, which was lowered when the filensin COOH-terminal peptide cRNA was coinjected with the cRNA for AQP0.
The filensin COOH-terminal tail region interacted with the AQP0 COOH-terminal region and the results strongly suggested that the interaction was direct. It appears that interactions between AQP0 and filensin helps to regulate the water permeability of AQP0 and to organize the structure of lens fiber cells, and may also help to maintain the transparency of the lens.
In contrast to many other organisms, the malarial parasite Plasmodium falciparum possesses only one typical glutathione S-transferase. This enzyme, PfGST, cannot be assigned to any of the known GST classes and represents a most interesting target for antimalarial drug development. The PfGST under native conditions forms non-covalently linked higher aggregates with major population (~98%) being tetramer. However, in the presence of 2 mM GSH, a dimer of PfGST is observed. Recently reported study on binding and catalytic properties of PfGST indicated a GSH dependent low-high affinity transition with simultaneous binding of two GSH molecules to PfGST dimer suggesting that GSH binds to low affinity inactive enzyme dimer converting it to high affinity functionally active dimer. In order to understand the role of GSH in tetramer-dimer transition of PfGST as well as in modulation of functional activity of the enzyme, detailed structural, functional and stability studies on recombinant PfGST in the presence and absence of GSH were carried out.
Our data indicate that the dimer – and not the tetramer – is the active form of PfGST, and that substrate saturation is directly paralleled by dissociation of the tetramer. Furthermore, this dissociation is a reversible process indicating that the tetramer-dimer equilibrium of PfGST is defined by the surrounding GSH concentration. Equilibrium denaturation studies show that the PfGST tetramer has significantly higher stability compared to the dimer. The enhanced stability of the tetramer is likely to be due to stronger ionic interactions existing in it.
This is the first report for any GST where an alteration in oligomeric structure and not just small conformational change is observed upon GSH binding to the enzyme. Furthermore we also demonstrate a reversible mechanism of regulation of functional activity of Plasmodium falciparum glutathione S-transferase via GSH induced dissociation of functionally inactive tetramer into active dimers.
Lymphatic filarial parasites survive within the lymphatic vessels for years despite the complex immune environment surrounding them. Parasites possibly accomplish this by adopting various immunomodulatory strategies, which include release of glutathione-S-transferases (GSTs) that counteract the oxidative free radicals produced by the host. Since GSTs produced by parasites appear to be critical for the survival of parasites in the host, several studies evaluated the potential of parasite GSTs as vaccine candidates especially against schistosomiasis, fascioliasis and Seteria cervi. However, vaccine potential of GSTs of lymphatic filarial parasites has not been evaluated before.
In the present study, the GST gene was cloned from the third stage larval (L3) cDNA libraries of Wuchereria bancrofti, and recombinant GST (WbGST) was expressed and purified. Serum samples from individuals living in an endemic area were analyzed for their reactivity with rWbGST. These findings showed that sera from endemic normal individuals (EN) carry significant levels of anti-WbGST IgG antibodies compared to subjects who are microfilaraemic (Mf) or show symptoms of clinical pathology (CP). Isotype analysis of the anti-WbGST IgG antibodies showed a predominance of IgG1 and IgG3 antibodies in EN individuals. Subsequent functional analysis of the rWbGST showed that the rWbGST protein retained the enzymatic activity of GST and the antibodies in EN sera could inhibit this enzymatic activity. Similar results were obtained when anti-rWbGST antibodies raised in mice were used in the neutralization assay. Brugia malayi GST and WbGST show significant sequence similarity. Therefore, to evaluate the vaccine potential of rWbGST, we used B. malayi L3 as challenge parasites. Vaccine potential of rWbGST was initially evaluated by confirming the role of human and mice WbGST antibodies in an antibody dependent cellular cytotoxicity (ADCC) assay. Subsequent vaccination studies in a jird model showed that approximately 61% protection could be achieved against a B. malayi L3 challenge infection in jirds immunized with rWbGST.
Results of this study show that rWbGST is a potential vaccine candidate against lymphatic filariasis. Nearly 61% protection can be achieved against a B. malayi challenge infection in a jird model. The study also showed that the WbGST protein retained the enzymatic activity of GST and this enzymatic activity appears to be critical for the survival of the parasite in the host.
Lymphatic parasites survive for years in a complex immune environment by adopting various strategies of immune modulation, which includes counteracting the oxidative free radical damage caused by the host. We now know that the filarial parasites secrete antioxidant enzymes. Among these, the glutathione-S-transferases (GSTs) have the potent ability to effectively neutralize cytotoxic products arising from reactive oxygen species (ROS) that attack cell membranes. Thus, GSTs have the potential to protect the parasite against host oxidative stress. GSTs of several helminthes, including schistosomes, fasciola and the filarial parasite Seteria cervi, are also involved in inducing protective immunity in the host. The schistosome 28 kDa GST has been successfully developed into a vaccine and is currently in Phase II clinical trials. Thus, GST appears to be a potential target for vaccine development. Therefore, in the present study, we cloned W. bancrofti GST, and expressed and purified the recombinant protein. Immunization and challenge experiments showed that 61% of protection could be achieved against B. malayi infections in a jird model. In vitro studies confirm that the anti-WbGST antibodies participate in the killing of B. malayi L3 through an ADCC mechanism and enzymatic activity of WbGST appears to be critical for this larvicidal function.
UL97 is a protein kinase encoded by human cytomegalovirus (HCMV) and is an important target for antiviral drugs against this ubiquitous herpesvirus, which is a major cause of life-threatening opportunistic infections in the immunocompromised host. In an effort to better understand the function(s) of UL97 during HCMV replication, a recombinant HCMV, NTAP97, which expresses a tandem affinity purification (TAP) tag at the amino terminus of UL97, was used to obtain UL97 protein complexes from infected cells. pp65 (also known as UL83), the 65-kDa virion tegument phosphoprotein, specifically copurified with UL97 during TAP, as shown by mass spectrometry and Western blot analyses. Reciprocal coimmunoprecipitation experiments using lysates of infected cells also indicated an interaction between UL97 and pp65. Moreover, in a glutathione S-transferase (GST) pull-down experiment, purified GST-pp65 fusion protein specifically bound in vitro-translated UL97, suggesting that UL97 and pp65 do not require other viral proteins to form a complex and may directly interact. Notably, pp65 has been previously reported to form unusual aggregates during viral replication when UL97 is pharmacologically inhibited or genetically ablated, and a pp65 deletion mutant was observed to exhibit modest resistance to a UL97 inhibitor (M. N. Prichard, W. J. Britt, S. L. Daily, C. B. Hartline, and E. R. Kern, J. Virol. 79:15494-15502, 2005). A stable protein-protein interaction between pp65 and UL97 may be relevant to incorporation of these proteins into HCMV particles during virion morphogenesis, with potential implications for immunomodulation by HCMV, and may also be a mechanism by which UL97 is negatively regulated during HCMV replication.
The glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes which protect against chemical injury. In contrast to mammals, GST expression in fish has not been extensively characterized, especially in the context of detoxifying waterborne pollutants. In the Northwestern United States, coho salmon (Oncorhynchus kisutch) are an important species of Pacific salmon with complex life histories that can include exposure to a variety of compounds including GST substrates. In the present study we characterized the expression of coho hepatic GST to better understand the ability of coho to detoxify chemicals of environmental relevance. Western blotting of coho hepatic GST revealed the presence of multiple GST-like proteins of approximately 24–26 kDa. Reverse phase HPLC subunit analysis of GSH affinity-purified hepatic GST demonstrated six major and at least two minor potential GST isoforms which were characterized by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI MS–MS) and Fourier transform-ion cyclotron resonance (FT-ICR) MS analyses. The major hepatic coho GST isoforms consisted of a pi and a rho-class GST, whereas GSTs representing the alpha and mu classes constituted minor isoforms. Catalytic studies demonstrated that coho cytosolic GSTs were active towards the prototypical GST substrate 1-chloro-2,4-dinitrobenzene, as well as towards ethacrynic acid and nitrobutyl chloride. However, there was no observable cytosolic GST activity towards the pesticides methyl parathion or atrazine, or products of oxidative stress, such as cumene hydroperoxide and 4-hydroxynonenal. Interestingly, coho hepatic cytosolic fractions had a limited ability to bind bilirubin, reflecting a potential role in the sequestering of metabolic by-products. In summary, coho salmon exhibit a complex hepatic GST isoform expression profile consisting of several GST classes, but may have a limited a capacity to conjugate substrates of toxicological significance such as pesticides and endogenous compounds associated with cellular oxidative stress.
Glutathione S-transferases; Coho salmon; Oncorhynchus kisutch; Pesticides; LC–MS/MS
AIM: To express the complete PreS region of HBV in E.coli with good solubility and stability, and to establish an effective method for purification of the recombinant PreS protein.
METHODS: The complete PreS region (PreS1 and PreS2) was fused into a series of tags including glutathione S-transferase (GST), dihydrofolate reductase (DHFR), maltose binding protein (MBP), 6×histidine, chitin binding domain (CBD), and thioredoxin, respectively. Expression of recombinant PreS fusion proteins was examined by SDS-PAGE analysis and confirmed by Western blot. Two fusion proteins, thio-PreS, and PreS-CBD, with desirable solubility and stability, were subjected to affinity purification and further characterization.
RESULTS: Recombinant PreS fusion proteins could be synthesized with good yields in E.coli. However, most of these proteins except for thio-PreS and PreS-CBD were vulnerable to degradation or insoluble as revealed by SDS-PAGE and Western blot. Thio-PreS could be purified by affinity chromatography with nickel-chelating sepharose as the matrix. However, some impurities were also co-purified. A simple freeze-thaw treatment yielded most of the thio-PreS proteins in solution while the impurities were in the precipitate. Purified thio-PreS protein was capable of inhibiting the binding of HBV virion to a specific monoclonal antibody against an epitope within the PreS1 domain.
CONCLUSION: Increased solubility and stability of the complete PreS region synthesized in E.coli can be achieved by fusion with the thioredoxin or the CBD tag. A simple yet highly effective method has been established for the purification of the thio-PreS protein. Purified thio-PreS protein likely assumes a native conformation, which makes it an ideal candidate for studying the structure of the PreS region as well as for screening antivirals.
Hepatitis B virus; PreS; Expression; Purification
The utility of recombinant Toxoplasma gondii surface antigen P22 for the detection of specific T. gondii antibodies in human sera was evaluated. Polymerase chain reaction was used to produce a 438-bp fragment of the P22 gene; the fragment corresponded to the amino acids predicted to be in the processed, native antigen. The fragment was subcloned into pGEX-2T and was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. The fusion protein was purified in a soluble form and was found to be recognized by sera from infected individuals in immunoblots and an enzyme-linked immunosorbent assay. Immunoglobulin G antibodies in sera from 31 acutely infected patients in general reacted more strongly to the fusion protein than did those in sera from 31 patients with the chronic infection. None of the sera from a panel of 26 seronegative controls reacted with the fusion protein was separated from the GST partner by cleavage with thrombin, it retained its immunoreactivity and its electrophoretic mobility in polyacrylamide gels was found to be similar to that of native P22. By a modification of the published method for purification of the foreign polypeptide from the GST carrier, the recombinant P22 was readily purified to homogeneity by thrombin cleavage of the fusion protein while it was adsorbed to glutathione agarose.
The 2-oxoglutarate-dependent dioxygenases (2ODDs) of gibberellin (GA) biosynthesis have a key role in the metabolism of a major plant hormone. The activity of recombinant GA 2ODDs from many species has been characterised in detail, however little information relates to enzyme purification. Native GA 2ODDs displayed lability during purification.
Two GA 2ODDs were expressed in Escherichia coli and purified to homogeneity. The GA 2-oxidase from Pisum sativum L., PsGA2OX1, was expressed as a glutathione s-transferase (GST) fusion. It was purified in the three steps of affinity chromatography, GST removal and gel filtration. Highly pure PsGA2OX1 was obtained at a yield of 0.3 mg/g of cells. It displayed a Km of 0.024 μM and a Vmax of 4.4 pkat/mg toward [1β,2β,3β-3H3]GA20. The GA 3-oxidase from Arabidopsis thaliana, AtGA3OX4, was expressed as a poly(His)-tagged thioredoxin fusion. It was purified by Immobilised Metal Affinity Chromatography followed by gel filtration. Cleavage of the fusion took place between the two purification steps. Highly pure AtGA3OX4 was obtained at a yield of 0.01 mg/g of cells. It displayed a Km of 0.82 μM and Vmax of 52,500 pkat/mg toward [1β,2β,3β-3H3]GA20.
Fusion tags were required to stabilise and solubilise PsGA2OX1 and AtGA3OX4 during E. coli expression. The successful purification of milligram quantities of PsGA2OX1 enables mechanistic and structural studies not previously possible on GA 2ODDs. A moderate yield of pure AtGA3OX4 requires the further optimisation of the latter stages of the enzyme purification schedule. PsGA2OX1's action in planta as deduced from the effect of the null mutation sln on GA levels in seeds is in agreement with the kinetic parameters of the recombinant enzyme.
In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes.
The cDNA coding for mouse fibroblast tropomyosin isoform 2 (TM2) was placed into a bacterial expression vector to produce a fusion protein containing glutathione-S-transferase (GST) and TM2 (GST/TM2). Glutathione-Sepharose beads bearing GST/TM2 were incubated with [35S]methionine-labeled NIH 3T3 cell extracts and the materials bound to the fusion proteins were analyzed to identify proteins that interact with TM2. A protein of 10 kD was found to bind to GST/TM2, but not to GST. The binding of the 10-kD protein to GST/TM2 was dependent on the presence of Ca2+ and inhibited by molar excess of free TM2 in a competition assay. The 10-kD protein-binding site was mapped to the region spanning residues 39-107 on TM2 by using several COOH-terminal and NH2-terminal truncation mutants of TM2. The 10-kD protein was isolated from an extract of NIH 3T3 cells transformed by v-Ha-ras by affinity chromatography on a GST/TM2 truncation mutant followed by SDS- PAGE and electroelution. Partial amino acid sequence analysis of the purified 10-kD protein, two-dimensional polyacrylamide gel analysis and a binding experiment revealed that the 10-kD protein was identical to a calcium-binding protein derived from mRNA named pEL98 or 18A2 that is homologous to S100 protein. Immunoblot analysis of the distribution of the 10-kD protein in Triton-soluble and -insoluble fractions of NIH 3T3 cells revealed that some of the 10-kD protein was associated with the Triton-insoluble cytoskeletal residue in a Ca(2+)-dependent manner. Furthermore, immunofluorescent staining of NIH 3T3 cells showed that some of the 10-kD protein colocalized with nonmuscle TMs in microfilament bundles. These results suggest that some of the pEL98 protein interacts with microfilament-associated nonmuscle TMs in NIH 3T3 cells.
This study was aimed to examine circadian variations of hepatic antioxidant components, including the Nrf2- pathway, the glutathione (GSH) system, antioxidant enzymes and metallothionein in mouse liver.
Methods and Results
Adult mice were housed in light- and temperature-controlled facilities for 2 weeks, and livers were collected every 4 h during the 24 h period. Total RNA was isolated, purified, and subjected to real-time RT-PCR analysis. Hepatic mRNA levels of Nrf2, Keap1, Nqo1 and Gclc were higher in the light-phase than the dark-phase, and were female-predominant. Hepatic GSH presented marked circadian fluctuations, along with glutathione S-transferases (GST-α1, GST-µ, GST-π) and glutathione peroxidase (GPx1). The expressions of GPx1, GST-µ and GST-π mRNA were also higher in females. Antioxidant enzymes Cu/Zn superoxide dismutase (Sod1), catalase (CAT), cyclooxygenase-2 (Cox-2) and heme oxygenase-1 (Ho-1) showed circadian rhythms, with higher expressions of Cox-2 and CAT in females. Metallothionein, a small non-enzymatic antioxidant protein, showed dramatic circadian variation in males, but higher expression in females. The circadian variations of the clock gene Brain and Muscle Arnt-like Protein-1(Bmal1), albumin site D-binding protein (Dbp), nuclear receptor Rev-Erbα (Nr1d1), period protein (Per1 and Per2) and cryptochrome 1(Cry1) were in agreement with the literature. Furthermore, acetaminophen hepatotoxicity is more severe when administered in the afternoon when hepatic GSH was lowest.
Circadian variations and gender differences in transcript levels of antioxidant genes exist in mouse liver, which could affect body responses to oxidative stress at different times of the day.
It has been suggested that hepatitis B virus (HBV) binds to a receptor on the plasma membrane of human hepatocytes via the pre-S1 domain of the large envelope protein as an initial step in HBV infection. However, the nature of the receptor remains controversial. In an attempt to identify a cell surface receptor for HBV, purified recombinant fusion protein of the pre-S1 domain of HBV with glutathione S-transferase (GST), expressed in Escherichia coli, was used as a ligand. The surface of human hepatocytes or HepG2 cells was biotinylated, and the cell lysate (precleared lysate) which did not bind to GST and glutathione-Sepharose beads was used as a source of receptor molecules. The precleared lysate of the biotinylated cells was incubated with the GST–pre-S1 fusion protein, and the bound proteins were visualized by Western blotting and enhanced chemiluminescence. An approximately 80-kDa protein (p80) was shown to bind specifically to the pre-S1 domain of the fusion protein. The receptor binding assay using serially or internally deleted segments of pre-S1 showed that amino acid residues 12 to 20 and 82 to 90 are essential for the binding of pre-S1 to p80. p80 also bound specifically to the pre-S1 of native HBV particles. Analysis of the tissue and species specificity of p80 expression in several available human primary cultures and cell lines of different tissue origin showed that p80 expression is not restricted to human hepatocytes. Taken together the results suggest that p80 may be a component of the viral entry machinery.