Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Vascular endothelial growth factor receptor 1 (Flt1) plays an important role in angiogenesis. It was hypothesized that, upon binding to VEGF, Flt1 tyrosine kinase underwent dimerization and initiated the signal transduction in VEGF/VEGF receptor system. In this report, a soluble active Flt1 tyrosine kinase domain expressed in E. coli was obtained, and its properties were partly characterized. The cDNA of Flt1 tyrosine kinase domain was obtained from the total RNA extracted from human liver cancer tissues by using RT-PCR, and was cloned to vector pGEX-KG. A soluble active GST-fusion protein of Flt1 tyrosine kinase domain (GST-F) was obtained from E. coli BL21 (DE3) pLysS. Although it was reported that GST-F contains no phosphorylation site, it did autophosphorylate in vitro. Mg2+ and Mn2+ were essential for the activity. It was also found that GST-F phosphorylated a synthesized substrate PolyE4Y, but not MBP and Src-related-peptide. The optimal Mg2+ and Mn2+ concentration for polyE4Y phosphorylation was 15 mmol/L and 0.5 mmol/L, respectively. This work is helpful for developing the new anti-cancer drugs.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2002 Jan
PMID:[Cloning, expression and characterization of human vascular endothelial growth factor receptor 1 tyrosine kinase]. 1195 32

A yeast two-hybrid screening by using PAP1 was performed to identify targets for PAP1-PHO85 cyclin-CDK complex. N-terminal fragment of protein YLR190w, a yeast gene encoding a 491 amino acids peptide, was identified, and its coding region was amplified by PCR. The interaction of PAP1 and YLR190w was confirmed by both two-hybrid assay and GST pull-down assay in vitro. The PAP1-PHO85 kinase complex obtained from the immunoprecipitates could phosphorylate GST-YLR190w expressed in E.coli, and the phosphorylation of YLR190w was affected by the phosphate concentration, and the phosphorylation sites of YLR190w were Ser/Thr-Promotif, as revealed by protein mutation assay. In another library screen, YAF9, a yeast homolog of human AF9, was isolated using the two-hybrid system with YLR190w as the bait. It was revealed that interaction of YLR190w and YAF9 was affected by phosphate concentration. When all Ser/Thr in Ser/Thr-Pro motif were mutated to Ala, the interaction of YLR190w (mutant) and YAF9 was weakened, and the effect of phosphate concentration was impaired. Ylr190w was not involved in the PHO system by the acid phosphatase activity assay. Deletion of Ylr190w was constructed by homologous recombination and the doubling time of Ylr190w mutant strain as longer than that of wild type.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2002 Mar
PMID:[Phosphorylation of YLR190w by PAP1 PHO85 kinase complex]. 1200 94

Connexin 31 is a member of connexin family. The carboxy-terminal cytosolic domain of connexin 31 contains several potential phosphorylation sites. In this work, a yeast two-hybrid protein interaction screen have been used to identify proteins that bind to the carboxy-terminus of connexin 31, and the p11 protein, an unique member of S100 protein family, and one of the two subunits of the annexin II tetramer was isolated. Interestingly, from yeast two-hybrid AD's coding sequence, three different reading frames of p11 DNA sequence were found,which come from different AD plasmids. By constructing AD plasmids using p11 ORF or 5' UTR, the protein coding by p11 ORF bind to connexin 31, while polypeptides coding by three kinds of 5 UTR did not bind to connexin 31, suggesting a translational frameshift of p11 fusion protein. To construct baits by dividing connexin 31 C-terminus into two domain, the p11 binding domain of connexin 31 was found located between 206-237 codons. The plasmid Cx31CT-pGEX-4T-2 was constructed for expression and purification of GST-Cx31CT; and p11-pQE30 for expression and purification of 6xHis-p11. In vitro binding assay showed that recombinant Cx31 interacted with recombinant p11.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2002 Mar
PMID:[Translational frameshift may be occur in p11, an interaction protein of Cx31, in yeast]. 1200

Tax is a transcription activator encoded by human T-cell leukemia virus (HTLV)-1. Ribosomal protein L6 was also defined as Taxreb107 (Tax responsible element binding protein 107) for its activity of binding to the long terminal repeats of HTLV-1. To investigate the relationship between Tax and Taxreb107/RpL6, yeast two hybrid and GST pull-down assays were used. Results suggest that Tax can interact with Taxreb107/RpL6 directly and Taxreb107/RpL6 may regulate the function of Tax in HTLV-1 proliferation.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2002 Mar
PMID:[Interaction between HTLV-1 transcription activator tax and Taxreb107]. 1200 2

Discoidin domain receptor 2 (DDR2) is a new type of receptor tyrosine kinases, and was thought to be involved in the metastasis of some tumors. Its ligand is fibrillar collagen. The activation of DDR2 induced by collagen mediates the over-expression of matrix metalloproteinase 1 (MMP-1) in cells. A specific inhibitor of DDR2 was necessary for the study of DDR2 function. Theoretically, a soluble receptor could possibly be used as specific inhibitor for the native receptor on cell membrane. In this report, a fragment (DB) of extracellular part of DDR2 was cloned and expressed for the use as potential inhibitor. This DB fragment corresponded to the polypeptide from the 23rd amino acid residue to the 293rd amino acid residue of DDR2. The fragment was amplified by RT-PCR from human lung cancer tissue, and the product was cloned into pMD18-T vector. After identification by sequence analysis, the fragment was sub-cloned into pGEX-4T-1 vector. Fusion protein of GST-DB was expressed in JM109 E.coli cells as expected and the soluble part accounted for about 13% of the total fusion protein. The soluble fusion protein was then purified with glutathione affinity resin, and GST-DB with purity of 86.1% was obtained. Competitive combination inhibitory test showed that the purified GST-DB inhibited the interaction between collagen II and DDR2 on the surface of RA synovial fibroblasts. Zymography analysis showed that the level of MMP-1 of both NIH 3T3 cell and RA synovial fibroblasts with collagen II-stimulation decreased after adding GST-DB fusion protein. The results indicated that the fusion protein GST-DB could inhibit the function of DDR2 on cells, and DDR2 might mediate collagen II-induced over-expression of MMP-1 in these cells.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2001
PMID:Expression, Purification and Functional Identification of Extracellular Part of Discoidin Domain Receptor 2. 1203 56

The efficacy of cytokine therapy has been demonstrated in several viral diseases. Interferon-gamma is a cytokine that has potent antiviral property and immunomodulatory activity. To investigate the role of IFN-gamma in viral clearance during natural infection and to define the antiviral mechanism, DHBV-infected ducks was used as an animal model. To clone, express, and develop the method of quantifying DuIFN-gamma gene transcription and expression, DuIFN -gamma cDNA was amplified by RT-PCR from PHA stimulated duck PBMC. Recombinant plasmid expressing DuIFN-gamma was used to transfect COS-7, and the cell culture supernatant was analyzed by CPE inhibitory assay and MTT methods to determine the antiviral titer of IFN-gamma. The GST-DuIFN-gamma fusion protein was expressed in E.coli and purified using the GST sepharose 4B. Results indicated that the supernatant collected from COS-7 cells transfected with DuIFN-gamma cDNA was able to prevent duck fibroblasts from VSV induced CPE in a dose dependent manner. An anti-DuIFN-gamma antibody neutralized this antiviral activity.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2001
PMID:Cloning and Expression of Chinese Duck Interferon-gamma Gene. 1203 67

Expression plasmids pGEXSI and pGEXSII containing one copy and two orderly joined copies of PreS1(21--47 aa) DNA fragment, respectively, were constructed. GST-PreS1(21--47 aa) and GST-2xPreS1(21--47 aa) fusion proteins were highly expressed in E.Coli TG1, induced by IPTG. The expression level of GST-PreS1(21--47 aa) was about 30% of total soluble proteins in the lysate of expression bacteria, and GST-2xPreS1(21--47 aa) was about 15% of total soluble proteins, asestimated by SDS-PAGE. 50 mg GST-PreS1(21--47 aa) or 20 mg GST-2xPreS1(21--47 aa) with purity over 90% was obtained, respectively, from 1 L culture by using affinity chromatography of glutathione-Sepharose 4B. Direct ELISA results showed that antigenicity of GST-2xPreS1(21--47 aa) was better than GST-PreS1(21--47 aa) and synthetic peptide. Using GST-2xPreS1(21--47 aa) as coated antigen, a sensitive indirect ELISA for detection of anti-PreS1(21--47 aa) antibody, based on protein A-biotin and streptavidin-HRP, was established. The results from 99 sera samples of hepatitis B patients showed that anti-PreS1(21--47 aa) antibody was detected in nearly half of acute hepatitis B patients during recovery, but it was detected only in a few chronic hepatitis patients. Clinical follow-up study suggested that appearance of anti-PreS1(21--47 aa) was related to the course of the disease and recovery of patients. Detection system established in the study is promising for clinical application.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2001
PMID:Expression, Purification and Preliminary Clinical Use of Recombinant HBsAg GST-PreS1(21--47 aa) Fusion Proteins. 1204 Apr 9

A cDNA for the disintegrin domain (hf279) was isolated by PCR from human testis cDNAs. DNA sequencing indicated that hf279 cDNA encoded 93 amino acid residues, and it was identical with the reported sequence of fertilin beta. An expression plasmid, pGEX hf279, was constructed by inserting hf279 cDNA into plasmid pGEX-4T-2 containing gst gene. The expression plasmid was introduced into E.coli BL21(DE3) cells and a substantial amount of soluble fused protein GST-HF93 was obtained by the expression strain HF93/BL21 induced with IPTG. SDS-PAGE analysis revealed that the GST-HF93 fusion protein had an apparent molecular weight of 38 kD and accumulated up to 50% of bacterial soluble proteins. The fusion protein was purified by glutathione S-transferase (GST) Sepharose 4B column (purity 90%) and digested by thrombin to obtain the purified HF93 peptide (purity 80%). Polyclonal antibodies were obtained from the serum of miceimmunized with purified HF93 which was isolated by GST Sepharose 4B column and SDS-PAGE. ELISA and Western blot analysis showed its specificity to HF93. Therefore this antibody can be used in further studies on the function of HF93.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2001
PMID:Cloning, Expression and Antibody Production of the Disintegrin Domain of Human Fertilin beta. 1205 Jul 90

The cloning and sequence analysis of Chinese human cardiac myosin light chain 1 (CCMLC1) was previously reported. In this paper the cDNA of CCMLC1 was used as template and both of cDNAs of N and C terminal fragments of CCMLC1, each containing 98 amino acid residues, were obtained by PCR. Using the expressed products of both fragments, the binding experiments of two fragments to cardiacmyosin heavy chain of rat, human cardiac actin and to monoclonal antibody raised against CCMLC1, have been performed, respectively, by means of precipitation with GST-Sepharose beads. The results showed that all the heavy chain, actin and monoclonal antibody bound the N terminal fragment of CCMLC1 at different sites. Under experimental conditions, the binding of CCMLC1 with actin could affect the subsequent binding of CCMLC1 to heavy chain in topologically.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2001
PMID:Binding of Human Cardiac Myosin Light Chain 1 to Heavy Chain and Actin. 1205 87

GST fusion protein expression system combined with protein truncation test(PTT) protocol was used to detect gene frame shift mutation. The RT-PCR products of Lis1 genes from hepatocarcinoma samples were respectively cloned into a GST fusion protein expression vector pGEX-1, then expressed in E.coli. The results showed a truncated 33 kD fusion protein in SDS-PAGE, although the full-translated product of Lis1 gene should be of 71 kD. Sequencing revealed insertion of an A residue, causing the premature termination of translation, between the 163th and 164th nucleotide of Lis1 gene. This improved PTT assay was proved to be a fast and effective way in detecting gene frame shift mutation.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2000
PMID:Detection of Lis1 Gene Frame Shift Mutation in Human Hepatocarcinoma. 1207 32


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