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1.
Int J Mol Sci ; 24(4)2023 Feb 12.
Article in English | MEDLINE | ID: mdl-36835112

ABSTRACT

Glutathione transferases (GSTs) are promiscuous enzymes whose main function is the detoxification of electrophilic compounds. These enzymes are characterized by structural modularity that underpins their exploitation as dynamic scaffolds for engineering enzyme variants, with customized catalytic and structural properties. In the present work, multiple sequence alignment of the alpha class GSTs allowed the identification of three conserved residues (E137, K141, and S142) at α-helix 5 (H5). A motif-directed redesign of the human glutathione transferase A1-1 (hGSTA1-1) was performed through site-directed mutagenesis at these sites, creating two single- and two double-point mutants (E137H, K141H, K141H/S142H, and E137H/K141H). The results showed that all the enzyme variants displayed enhanced catalytic activity compared to the wild-type enzyme hGSTA1-1, while the double mutant hGSTA1-K141H/S142H also showed improved thermal stability. X-ray crystallographic analysis revealed the molecular basis of the effects of double mutations on enzyme stability and catalysis. The biochemical and structural analysis presented here will contribute to a deeper understanding of the structure and function of alpha class GSTs.


Subject(s)
Glutathione Transferase , Isoenzymes , Humans , Models, Molecular , Glutathione Transferase/genetics , Isoenzymes/metabolism , Catalysis , Kinetics , Glutathione , Binding Sites
2.
Int J Mol Sci ; 22(5)2021 Feb 26.
Article in English | MEDLINE | ID: mdl-33652971

ABSTRACT

Drought and heat stresses are major factors limiting crop growth and productivity, and their effect is more devastating when occurring concurrently. Plant glutathione transferases (GSTs) are differentially expressed in response to different stimuli, conferring tolerance to a wide range of abiotic stresses. GSTs from drought-tolerant Phaseolus vulgaris var. "Plake Megalosperma Prespon" is expected to play an important role in the response mechanisms to combined and single heat and drought stresses. Herein, we examined wild-type N. tabacum plants (cv. Basmas Xanthi) and T1 transgenic lines overexpressing the stress-induced Pvgstu3-3 and Pvgstu2-2 genes. The overexpression of Pvgstu3-3 contributed to potential thermotolerance and greater plant performance under combined stress. Significant alterations in the primary metabolism were observed in the transgenic plants between combined stress and stress-free conditions. Stress-responsive differentially expressed genes (DEGs) and transcription factors (TFs) related to photosynthesis, signal transduction, starch and sucrose metabolism, osmotic adjustment and thermotolerance, were identified under combined stress. In contrast, induction of certain DEGs and TF families under stress-free conditions indicated that transgenic plants were in a primed state. The overexpression of the Pvgstu3-3 is playing a leading role in the production of signaling molecules, induction of specific metabolites and activation of the protective mechanisms for enhanced protection against combined abiotic stresses in tobacco.


Subject(s)
Gene Expression Regulation, Plant , Nicotiana/genetics , Droughts , Genes, Plant , Hot Temperature , Plant Proteins/genetics , Stress, Physiological , Thermotolerance , Nicotiana/physiology
3.
Fish Shellfish Immunol ; 82: 1-8, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30064015

ABSTRACT

The microalgae are an important source of bioactive molecules including ß-glucans that can be used as immunostimulants in aquaculture. In the present study, the antioxidant capacity, cytotoxicity and immunomodulatory activity of a chrysolaminarin-enriched extract obtained from the diatom Phaeodactylum tricornutum was evaluated. The extract showed a higher total antioxidant activity as determined by ORAC and FRAP assays and a lower DPPH scavenging activity than particulate yeast-ß-glucan. The cytotoxicity test indicated that extract concentrations higher than 0.01% w/v could impair cell viability of human dermal fibroblasts. To evaluate the immunomodulatory activity, juvenile soles were intraperitoneally injected with the chrysolaminarin-enriched extract suspended in coconut oil (1 mg/fish) followed by a reinjection at 7 days. A sham group injected with the carrier solution was maintained as a negative control. Cumulated mortality of fish injected with the chrysolaminarin-enriched extract was 29.4% after six days and no mortality was recorded after extract reinjection. Expression analyses of fifteen genes related to the innate immune system in kidney, spleen and intestine showed temporal and organ-specific responses. A rapid (2 days post-injection; dpi) and strong induction of the pro-inflammatory il1b and the antimicrobial peptide hamp1 in the three immunological organs, the hsp90aa in kidney and spleen, irf3 in intestine and c3 in spleen was observed indicating a potent inflammatory response. The recovery of steady-state levels for all activated genes at 5 dpi, and the down-regulation of c-lectin receptor as well as some interferon-related genes (ifn1, irf1, irf3, irf8, irf9 and mx) in kidney and cxc10 in spleen indicated that the soles were able to activate a homeostatic response against the ß-glucan insult. The reinjection of the chrysolaminarin-enriched extract did not activate a new inflammatory response but reduced the mRNA levels of hsp90aa and irf3 indicating that soles developed some resistance to ß-glucans. Overall, these results reveal this enriched extract as a novel and potent source of ß-glucans with antioxidant and immunomodulatory capacity suitable for immunostimulation in aquaculture.


Subject(s)
Adjuvants, Immunologic/pharmacology , Antioxidants/pharmacology , Diatoms/chemistry , Fish Diseases/immunology , Flatfishes , Immunity, Innate/drug effects , Animals , Fish Diseases/genetics , Gene Expression Profiling/veterinary , Immunity, Innate/genetics , Injections, Intraperitoneal/veterinary , Microalgae/chemistry
4.
Biochim Biophys Acta Gen Subj ; 1861(1 Pt A): 3416-3428, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27612661

ABSTRACT

BACKGROUND: Glutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. METHODS: A library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). RESULTS: Activity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher kcat/Km and improved thermal stability, compared to the parent enzymes. The crystal structures of the GSTD4 enzyme in free form and in complex with GSH were determined to 1.6Å and 2.3Å resolution, respectively. Analysis of the GSTD4 structure showed subtle conformational changes in the GSH-binding site and in electron-sharing network that may contribute to the increased GSH affinity. The shuffled variant GSTD4 was further optimized for improved oxidative stability employing site-saturation mutagenesis. The Cys112Ser mutation confers optimal oxidative stability and kinetic properties in the GSTD4 enzyme. CONCLUSIONS: DNA shuffling allowed the creation of a chimeric enzyme variant with improved properties, compared to the parent enzymes. X-ray crystallography shed light on how recombination of a specific segment from homologous GSTA1-1 together with point mutations gives rise to a new functionally competent enzyme with improved binding, catalytic properties and stability. GENERAL SIGNIFICANCE: Such an engineered GST would be useful in biotechnology as affinity tool in affinity chromatography as well as a biocatalytic matrix for the construction of biochips or enzyme biosensors.


Subject(s)
DNA Shuffling , Glutathione Transferase/metabolism , Glutathione/metabolism , Isoenzymes/metabolism , Adsorption , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , DNA, Complementary/genetics , Enzyme Activation , Enzyme Stability , Glutathione Transferase/chemistry , Humans , Isoenzymes/chemistry , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Oxidation-Reduction , Rats , Temperature
5.
Methods Mol Biol ; 2178: 201-215, 2021.
Article in English | MEDLINE | ID: mdl-33128752

ABSTRACT

Dye-ligand affinity chromatography is a widely used technique in protein purification. The utility of the reactive dyes as affinity ligands results from their unique chemistry, which confers wide specificity toward a large number of proteins. They are commercially available, inexpensive, stable and can easily be immobilized. Significant factors that contribute to the successful operation of a dye-ligand chromatography include matrix type, dye-ligand density, adsorption along with elution conditions and flow rate. The present chapter provides protocols for the synthesis of dye-ligand affinity adsorbents as well as protocols for screening, selection, and optimization of a given dye-ligand purification step. The purification of the glutathione transferases from Phaseolus vulgaris on Cibacron Blue 3GA-Sepharose affinity adsorbent is given as an example.


Subject(s)
Glutathione Transferase , Phaseolus/enzymology , Plant Proteins , Sepharose/analogs & derivatives , Glutathione Transferase/chemistry , Glutathione Transferase/isolation & purification , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Sepharose/chemistry
6.
Methods Mol Biol ; 2089: 41-46, 2020.
Article in English | MEDLINE | ID: mdl-31773646

ABSTRACT

Drug development is the process of bringing a new pharmaceutical drug to the market once a lead compound has been identified through the process of drug discovery. Enzymes are one of the most important groups of drug targets; thus, enzyme inhibition is widely used for the treatment of certain disorders. The assessment of an inhibitor against an enzyme is predominantly based on two different parameters: the half-maximal inhibitory concentration (IC50) and the inhibition constant (Ki). This chapter describes an experimental procedure for the determination of the IC50 value of an enzyme inhibitor. The relationship between IC50 and Ki is also discussed.


Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Inhibitory Concentration 50 , Kinetics , Molecular Docking Simulation , Pharmaceutical Preparations/chemistry
7.
Methods Mol Biol ; 2089: 235-243, 2020.
Article in English | MEDLINE | ID: mdl-31773658

ABSTRACT

Ligand fishing is a convenient bioanalytical screening method that is based on the affinity selection of a ligand from a complex biological sample by an immobilized receptor. It is a versatile affinity-based screening approach and it has found application in multiple interacting pairs such as enzyme-inhibitor/activator, antigen-antibody, receptor-ligand, and protein-protein. Important parameters that affect the successful operation of the method are the high specificity and strong binding affinity of the interacting pair (e.g., enzyme-ligand complex) and the elution of the bound ligand from the complex. This chapter provides protocols for the synthesis of affinity adsorbent and its application in off-line ligand-fishing procedure for a 6His-tagged glutathione transferase (GST).


Subject(s)
Biological Products/chemistry , Biological Products/pharmacology , Antibodies/metabolism , Chromatography, Affinity/methods , Drug Discovery/methods , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/pharmacology , Glutathione Transferase/metabolism , Ligands , Protein Binding/physiology , Proteins/metabolism
8.
Curr Pharm Des ; 26(40): 5205-5212, 2020.
Article in English | MEDLINE | ID: mdl-32713331

ABSTRACT

BACKGROUND: Glutathione transferases (GSTs) are a family of Phase II detoxification enzymes that have been shown to be involved in the development of multi-drug resistance (MDR) mechanism toward chemotherapeutic agents. GST inhibitors have, therefore, emerged as promising chemosensitizers to manage and reverse MDR. Colchicine (COL) is a classical antimitotic, tubulin-binding agent (TBA) which is being explored as anticancer drug. METHODS: In the present work, the interaction of COL and its derivative 2,3-didemethylcolchicine (2,3-DDCOL) with human glutathione transferases (hGSTA1-1, hGSTP1-1, hGSTM1-1) was investigated by inhibition analysis, molecular modelling and molecular dynamics simulations. RESULTS: The results showed that both compounds bind reversibly to human GSTs and behave as potent inhibitors. hGSTA1-1 was the most sensitive enzyme to inhibition by COL with IC50 22 µΜ. Molecular modelling predicted that COL overlaps with both the hydrophobic (H-site) and glutathione binding site (G-site) and polar interactions appear to be the driving force for its positioning and recognition at the binding site. The interaction of COL with other members of GST family (hGSTA2-2, hGSTM3-3, hGSTM3-2) was also investigated with similar results. CONCLUSION: The results of the present study might be useful in future drug design and development efforts towards human GSTs.


Subject(s)
Antineoplastic Agents , Colchicine , Antineoplastic Agents/pharmacology , Glutathione , Glutathione Transferase , Humans , Microtubules
9.
Biochim Biophys Acta Gen Subj ; 1863(3): 565-576, 2019 03.
Article in English | MEDLINE | ID: mdl-30590099

ABSTRACT

In the present work, a structure-based design approach was used for the generation of a novel variant of synthetic glutathione transferase (PvGmGSTU) with higher sensitivity towards pesticides. Molecular modelling studies revealed Phe117 as a key residue that contributes to the formation of the hydrophobic binding site (H-site) and modulates the affinity of the enzyme towards xenobiotic compounds. Site-saturation mutagenesis of position Phe117 created a library of PvGmGSTU variants with altered kinetic and binding properties. Screening of the library against twenty-five different pesticides, showed that the mutant enzyme Phe117Ile displays 3-fold higher catalytic efficiency and exhibits increased affinity towards α-endosulfan, compared to the wild-type enzyme. Based on these catalytic features the mutant enzyme Phe117Ile was explored for the development of an optical biosensor for α-endosulfan. The enzyme was entrapped in alkosixylane sol-gel system in the presence of two pH indicators (bromocresol purple and phenol red). The sensing signal was based on the inhibition of the sol-gel entrapped GST, with subsequent decrease of released [H+] by the catalytic reaction, measured by sol-gel entrapped indicators. The assay response at 562 nm was linear in the range pH = 4-7. Linear calibration curves were obtained for α-endosulfan in the range of 0-30 µΜ. The reproducibility of the assay response, expressed by relative standard deviation, was in the order of 4.1% (N = 28). The method was successfully applied to the determination of α-endosulfan in real water samples without sample preparation steps.


Subject(s)
Biosensing Techniques/methods , Environmental Monitoring/methods , Glutathione Transferase/chemistry , Glutathione Transferase/metabolism , Pesticides/analysis , Protein Engineering/methods , Binding Sites/genetics , Catalysis , Catalytic Domain/genetics , Directed Molecular Evolution/methods , Endosulfan/analysis , Endosulfan/isolation & purification , Environmental Pollutants/analysis , Environmental Pollutants/isolation & purification , Enzymes, Immobilized/genetics , Enzymes, Immobilized/metabolism , Glutathione/metabolism , Glutathione Transferase/genetics , Mutagenesis, Site-Directed , Mutant Proteins/metabolism , Pesticides/isolation & purification , Phaseolus/enzymology , Phaseolus/genetics , Reproducibility of Results , Glycine max/enzymology , Glycine max/genetics , Structure-Activity Relationship
10.
PLoS One ; 14(12): e0225666, 2019.
Article in English | MEDLINE | ID: mdl-31805094

ABSTRACT

The hatching enzymes or choriolysins are key proteases in fish life cycle controlling the release of larvae to surrounding environment that have been suggested as target for novel biotechnological uses. Due to the large amounts of eggs released by the flatfish Solea senegalensis, during the spawning season, the hatching liquid properties and choriolysin-encoding genes were investigated in this species. A genomic analysis identified four putative genes referred to as SseHCEa, SseHCEb, SseLCE and SseHE. The phylogenetic analysis classified these paralogs into two clades, the clade I containing SseHCE paralogs and the clade II containing two well-supported subclades named as HE and LCE. The two SseHCE paralogs were intron-less and both genes were tandemly arrayed very close in the genome. The synteny and gene rearrangement identified in the flatfish lineage indicated that the duplication of these two paralogs occurred recently and they are under divergent evolution. The genes SseHE and SseLCE were structured in 8 exons and 7 introns and the synteny was conserved in teleosts. Expression studies confirmed that the four genes were expressed in the hatching gland cells and they migrate co-ordinately from the head to around the yolk sac close to the hatch with specific temporal and intensity expression profiles. Although the mRNA levels of the four genes peaked in the hours previous to larval hatching, the SseHCE and SseLCE paralogs kept a longer expression than SseHE after hatching. These expression patterns were consistent even when larvae were incubated at different temperatures that modified hatching times. The analysis of hatching-liquid using SDS-PAGE and zymography analyses of hatching liquid identified a major band of expected choriolysin size. The optimal pH for protease activity was 8.5 and inhibition assays using EDTA demonstrated that most of the activity in the hatching liquid was due to metalloproteases with Ca2+ ions acting as the most effective metal to restore the activity. All these data provide new clues about the choriolysin evolution and function in flatfish with impact in the aquaculture and the blue cosmetic industry.


Subject(s)
Evolution, Molecular , Flatfishes/metabolism , Metalloendopeptidases , Animals , Flatfishes/genetics , Gene Expression Regulation, Developmental , Larva/genetics , Metalloendopeptidases/classification , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Phylogeny , RNA, Messenger/genetics
11.
12.
Curr Pharm Biotechnol ; 19(11): 925-931, 2018.
Article in English | MEDLINE | ID: mdl-30370843

ABSTRACT

BACKGROUND: Glutathione transferases (GSTs) catalyze the conjugation of glutathione (GSH) to endogenous and xenobiotic electrophilic compounds and have been involved in the development of resistance toward cancer chemotherapeutic drugs and in the etiology, pathology and progression of several other diseases. In the present work, the human isoenzyme GSTA1-1 (hGSTA1-1) was used to assemble a microplate-based platform for high-throughput screening of natural productbased inhibitors from plant extracts. METHODS: The enzyme was immobilized using sol-gel chemistry and deposited as a layer at the bottom surface of 96-well format ELISA microplate. The sensing signal was based on the inhibition of the colorimetric reaction between 1-chloro-dinitrobenzene (CDNB) and GSH, catalyzed by the sol-gel entrapped enzyme. RESULTS: As a proof of concept, the system was used for screening aqueous extracts from medicinal and aromatic plants with excellent reproducibility (approximately 95%). CONCLUSION: The operational simplicity and accuracy of this system, suggest that it can be explored as a bioanalytical tool with potential use in drug design and development efforts for finding new sources of GST inhibitors useful in chemomodulation of cancer drugs.


Subject(s)
Enzyme Inhibitors/pharmacology , Enzymes, Immobilized/antagonists & inhibitors , Glutathione Transferase/antagonists & inhibitors , Plant Extracts/pharmacology , Drug Design , Drug Evaluation, Preclinical , Enzyme Inhibitors/isolation & purification , Enzymes, Immobilized/chemistry , Glutathione Transferase/chemistry , High-Throughput Screening Assays , Humans , Plant Extracts/isolation & purification , Plants, Medicinal/chemistry , Reproducibility of Results
13.
Front Plant Sci ; 9: 1737, 2018.
Article in English | MEDLINE | ID: mdl-30555496

ABSTRACT

Glutathione transferases (GSTs, EC. 2.5.1.18) are inducible multifunctional enzymes that are essential in the detoxification and degradation of toxic compounds. GSTs have considerable biotechnological potential. In the present work, a new method for the generation of synthetic GSTs was developed. Abiotic stress treatment of Phaseolus vulgaris and Glycine max plants led to the induction of total GST activity and allowed the creation of a GST-enriched cDNA library using degenerated GST-specific primers and reverse transcription-PCR. This library was further diversified by employing directed evolution through DNA shuffling. Activity screening of the evolved library led to the identification of a novel tau class GST enzyme (PvGmGSTUG). The enzyme was purified by affinity chromatography, characterized by kinetic analysis, and its structure was determined by X-ray crystallography. Interestingly, PvGmGSTUG displayed enhanced glutathione hydroperoxidase activity, which was significantly greater than that reported so far for natural tau class GSTs. In addition, the enzyme displayed unusual cooperative kinetics toward 1-chloro-2,4-dinitrochlorobenzene (CDNB) but not toward glutathione. The present work provides an easy approach for the simultaneous shuffling of GST genes from different plants, thus allowing the directed evolution of plants GSTome. This may permit the generation of new synthetic enzymes with interesting properties that are valuable in biotechnology.

14.
Int J Biol Macromol ; 94(Pt B): 802-812, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27103493

ABSTRACT

The plant tau class glutathione transferases (GSTs) perform diverse catalytic as well as non-catalytic roles in detoxification of xenobiotics, prevention of oxidative damage and endogenous metabolism. In the present work, the tau class isoenzyme GSTU2-2 from Glycine max (GmGSTU2-2) was characterized. Gene expression analysis of GmGSTU2 suggested a highly specific and selective induction pattern to osmotic stresses, indicating that gene expression is controlled by a specific mechanism. Purified, recombinant GmGSTU2-2 was shown to exhibit wide-range specificity towards xenobiotic compounds and ligand-binding properties, suggesting that the isoenzyme could provide catalytic flexibility in numerous metabolic conditions. Homology modeling and phylogenetic analysis suggested that the catalytic and ligand binding sites of GmGSTU2-2 are well conserved compared to other tau class GSTs. Structural analysis identified key amino acid residues in the hydrophobic binding site and provided insights into the substrate specificity of this enzyme. The results established that GmGSTU2-2 participates in a broad network of catalytic and regulatory functions involved in the plant stress response.


Subject(s)
Glutathione Transferase/chemistry , Glutathione/chemistry , Glycine max/enzymology , Plant Proteins/chemistry , Seedlings/enzymology , tau Proteins/chemistry , Amino Acid Sequence , Cloning, Molecular , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Glutathione/metabolism , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Kinetics , Molecular Docking Simulation , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Domains , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Seedlings/chemistry , Seedlings/genetics , Sequence Alignment , Glycine max/chemistry , Glycine max/genetics , Stress, Physiological , Structural Homology, Protein , Substrate Specificity , Thermodynamics , tau Proteins/genetics , tau Proteins/metabolism
15.
Curr Pharm Biotechnol ; 18(11): 890-899, 2017.
Article in English | MEDLINE | ID: mdl-29278211

ABSTRACT

BACKGROUND: The starting point for the development of new, functional products derived from Rubus fruticosus L. is to determine the optimal cultivation conditions that produce maximal yield of fruits containing desirable bioactive properties. Towards that goal, the effect of soil, soil/peat mixture and light intensity on the nutraceutical and cosmeceutical potential of two cultivars ('Thornfree' and 'Loch Ness') of Rubus fruticosus L. were evaluated. METHODS: The assessment was carried out employing a range of methods for evaluating fruit properties associated with promoting good health such as total antioxidant capacity, secondary metabolites content (vitamin C, polyphenols, flavonoids and anthocyanins) and inhibition analysis of skin-regulating enzymes. RESULTS: 'Thornfree' cultivar produced fruits in all light conditions, while 'Loch Ness' did not produce fruits in low light conditions. The results showed that in Rubus fruticosus L. fruit, the chemical composition and bioactivity are strongly affected by both genetics factors and growing conditions. Extract from 'Thornfree' fruits obtained under low light and soil/peat conditions displayed superior properties such as high antioxidant capacity, high concentrations of phenolics, flavonoids and anthocyanins and high inhibitory potency towards the enzymes tyrosinase and elastase. This extract was used for the development of a topical skin care cream with excellent compatibility and stability. CONCLUSION: Our findings conclude that Rubus fruticosus L. cultivation may be efficiently and effectively manipulated through conventional cultivation techniques to produce promising bioactive ingredients with potential use in commercial cosmetics and pharmaceuticals.


Subject(s)
Agriculture/methods , Cosmeceuticals/analysis , Dietary Supplements/analysis , Fruit/chemistry , Plant Extracts/analysis , Rubus/physiology , Antioxidants/analysis , Ascorbic Acid/analysis , Flavonoids/analysis , Light , Phenols/analysis , Soil
16.
Curr Protoc Protein Sci ; Chapter 26: 26.6.1-26.6.10, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21400692

ABSTRACT

This unit describes a method for site-saturation mutagenesis (SSM) using PCR amplification with degenerate synthetic oligonucleotides as primers. SSM allows the substitution of predetermined protein sites against all twenty possible amino acids at once. Therefore, SSM is a powerful approach in protein engineering to characterize structure-function relationships, as well as to create improved protein variants. The procedure accepts double-stranded plasmid isolated from the dam(+) E. coli strain. The procedure is simple, fast, efficient, and eliminates time-consuming subcloning and ligation steps.


Subject(s)
Gene Library , Mutagenesis, Site-Directed/methods , Combinatorial Chemistry Techniques , Mutation , Polymerase Chain Reaction/methods , Protein Engineering/methods
17.
Recent Pat Biotechnol ; 3(3): 211-23, 2009.
Article in English | MEDLINE | ID: mdl-19747150

ABSTRACT

Cytosolic glutathione transferases (GSTs) are a diverse family of enzymes involved in a wide range of biological processes, many of which involve the conjugation of the tripeptide glutathione (GSH) to an electrophilic substrate. Detailed studies of GSTs are justified because of the considerable interest of these enzymes in medicine, agriculture and analytical biotechnology. For example, in medicine, GSTs are explored as molecular targets for the design of new anticancer drugs as a plausible means to sensitize drug-resistant tumors that overexpress GSTs. In agriculture, GSTs are exploited in the development of transgenic plants with increased resistance to biotic and abiotic stresses. Recently, selected isoenzymes of GSTs have found successful applications in the development of enzyme biosensors for the direct monitoring of environmental pollutants, such as herbicides and insecticides. This review article summarizes recent representative patents related to GSTs and their applications in biotechnology.


Subject(s)
Biotechnology/methods , Glutathione Transferase/genetics , Animals , Biosensing Techniques , Glutathione Transferase/chemistry , Glutathione Transferase/metabolism , Herbicide Resistance , Humans , Medicine , Plants, Genetically Modified
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