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)

To set up an in vitro amidating system, a recombinant human calcitonin with a glycine at C termial (mhCT-Gly) was used as the amidating substrate of recombinant rat peptidylglycine alpha-amidating monooxygenase (rPAM). First, the mhCT-Gly gene was synthesized and cloned into a fusion expression vector to get an expression plasmid pGEXCT. The GST-fused mhCT-Gly was highly expressed in E.coli BL21(DE3) harboring the pGEXCT, and was purified rapidly by affinity chromatography. Second, using the method of ultrafiltration, the rPAM was prepared from the supernatant of cultured transfectant CHO cells which express rPAM stably. Finally, the in vitro amidating experiments were carried out using GST-mhCT-Gly as substrate and the prepared rPAM. The results of dot blot with the specific antibody and of mass spectrum assay indicated that amidating product GST-hCT-NH(2) could be easily detected. This study provides a useful method for the amidation of recombinant products in vitro.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2000
PMID:In Vitro Amidating Processing of Products Expressed by Gene Engineering. 1207 61

PCL6, PCL7(PAP1), and PHO80 belong to the PHO80 subfamily of PCLs (PHO85 cyclins), share high homology in protein sequences, and function with some similarity. YLR190w, the substrate of PCL7-PHO85, shares homology with YJL084c in a 140-amino-acid region. In addition, YJL084c was reported as a PCL6-binding protein. Here, it was found that there was association between YJL084c and PCL7, and their interaction was confirmed by co-immunoprecipitation assay and GST pull-down assay. The in vitro translational product of YJL084c could be phosphorylated by PCL7-PHO85 complex. Also, the GST fusion protein of the middle region expressed in E.coli could be phosphorylated, while the amino terminal or the carboxyl terminal could not. Interestingly, PCL6-PHO85 complex had the same characters; effect of phosphate condition on the phosphorylation was shown in both PCL6-PHO85 and PCL7-PHO85. YPH499: Yjl084c :: LEU2 was constructed by homologous recombination. PUT4 was reported as a YJL084c-binding protein, but no difference was observed between wild strain and the Yjl084c null mutant on MP medium. In addition, the interaction between PHO81 and all of the three cyclins was analyzed.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2002 Jul
PMID:Analysis of phosphorylation of YJL084c, a yeast protein. 1209 64

An engineering E.coli strain, BL21 (DE3)/pGEX-4T hPTH (1-34), was constructed by oligonucleotide annealing and PCR amplifying the target gene, then ligating it with pGEX-4T-3 vector and transferring into BL21 host. The yield of soluble fusion protein of GST-hPTH(1-34) expressed from BL21(DE3)/pGEX-4T hPTH(1-34) is about 10 g/L after high-density, high expression culture and purification by affinity chromatography. Following the simple digestion of enterokinase, about 0.6 g/L intact hPTH (1-34) was harvested. The product is checked by HPLC MS and N-terminus sequence analysis. The purified recombinant hPTH(1-34) stimulated adenylate cyclase in rabbit renal cortical cell membranes to exactly the same extent as synthetic human parathyroid hormone standards, indicating that the recombinant product has full biological activity.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2002 Jul
PMID:Enterokinase cleavage of fusion proteins for preparation of recombinant human parathyroid hormone 1-34. 1209 70

The CtxB gene encoding cholerae toxin subunit B was amplified from Vibrio cholerae genomic DNA by PCR. The result of sequencing indicated that CtxB gene encodes 124 amino acid residues. The sequence of CtxB gene was almost the same as that of reported except for the codon of Thr 62. The expression plasmid pGEX-CTXB was constructed by inserting CtxB gene into plasmid pGEX-4T-2, containing gst gene, immediately downstream of the T7 promoter. The expressed plasmid was introduced into E.coli BL21(DE3) cells and expression strain CTXB/BL21 was selected. SDS-PAGE analysis revealed that the GST-CTXB fusion protein was highly expression and accumulated up to 36% of bacterial soluble proteins after the induction by IPTG. A fusion protein of 40 kD was expressed as inclusion body. The fusion protein was refolded and purified. The purified fusion protein was cut by thrombin to obtain the purified CTXB protein.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2000
PMID:Cloning of the CtxB Gene of Vibrio cholerae and Its Expression in E.coli. 1209 92

Human B1F was cloned from a human liver cDNA library by yeast one-hybrid screening and characterized. It is a transcription factor which belongs to nuclear receptor superfamily. The cDNA segment 450-930 of human liver transcription factor hB1F was cloned into the pGEX-3X expression vector and was expressed in E.coli. The purified GST-fused hB1F, named GST-CS, were used to immunize mice to get polyclonal antibodies to hB1F. The antiserum showed specificity to hB1F after the purification by GST-Sepharose 4B. It can be used in further investigations of the structure and function of hB1F.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2000
PMID:Fusion Expression and Antibody Preparation of the Human Transcription Factor hB1F. 1211 Sep 6

To reveal the possible role of the amino-terminal domain of G protein-coupled receptor kinases(GRKs)in receptor phosphorylation and/or modulation of its kinase activity, a truncated mutant of GRK-2 lacking the amino-terminal domain(deltaN-GRK2)was made. deltaN-GRK2 was expressed effectively in E.coli as a GST fusion protein and was purified by affinity chromatography on a GSH-Sepharose column. deltaN-GRK2 was then separated from GST tag by thrombin cleavage and recovered. Although deltaN-GRK2 had nearly identical activity with wild-type GRK-2 in phosphorylation of peptide substrate, it completely lost the ability to phosphorylate the light-activated receptor rhodopsin. Furthermore, deletion of the amino-terminal domain rendered GRK-2 unresponsive to the regulation of kinase activity by a truncated form of rhodopsin, (329)G-Rho(*) and beta gamma subunits of G protein. These results demonstrated that the amino-terminal domain was necessary to GRK2 for both the phosphorylation of receptor and the regulation of its kinase activity by the receptor. It was reasonable to postulate that this domain has little, if any effect on the catalytic domain of natural form of GRK2.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 1999
PMID:Effects of Deleting the Amino-terminal Domain of GRK-2 on Its Function. 1211 Sep 29

There are two kinds of glutathione peroxidases in cells, selenium-dependent glutathione peroxidase (SeGPx) and non-selenium-dependent glutathione peroxidase (non-SeGPx, GST), playing important roles in protecting cells from oxidative injury. In order to find out whether the effect of polysaccharide Krestin (PSK) is somehow associated with antioxidant enzymes, its effect on peroxidases gene expression was studied by determining glutathione peroxidase activities and the contents of glutathione peroxidase mRNA in PSK-treated mouse peritoneal macrophages. The results showed that PSK could improve glutathione peroxidase activities and increase the contents of glutathione peroxidase mRNA in mouse peritoneal macrophages; PSK might also induce the glutathione peroxidases expression through synthesis of a new protein.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 1999
PMID:The Effect of Polysaccharide Krestin on GPx Gene Expression in Macrophages. 1213 79

Applying real time BIA(biomolecular interaction analysis) the interaction between yeast PHO4 and PHO2 protein was analyzed. Recombinant PHO4 protein was coupled at the sensor chip via amino group. 5 &mgr;mol/L of recombinant PHO2 and PHO2 mutants which were fused with glutathione S-transferase were injected respectively. The mutant whose Ser 230 was changed into Asp (GST-2SD) showed high SPR (surface plasmon resonance) signal while wild type PHO2 and the mutant where Ser 230 was changed into Ala (GST-2SA) did not. This result indicated that there was interaction between PHO4 and GST-2SD proteins and the phosphorylation of the Ser 230 of PHO2 was essential. Five different concentration of GST-2SD (0.5 0.75 1.0 1.5 2.0&mgr;mol/L) were injected separately and the interaction kinetics was analyzed. The association rate constant is 2.4x10(4)(mol/L)(-1)s(-1) dissociation rate constant is 3.5x10(-5)s(-1) and association constant is 6.9x10(8) (mol/L)(-1).
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 1999
PMID:Analysis of Interaction between PHO4 and PHO2 Protein by Real Time BIA. 1213 4

There are six ankyrin repeats in yeast transcriptional factor PHO81. The PHO81 ankyrin repeats fused with glutathione S-transferase (GST-ANK) was overexpressed in E. coli and it existed in inclusion body form. Using reduced and oxidized glutathione systems the fused protein fragment was denatured and renatured and the soluble protein was obtained. Then it was purified by affinity purification through glutathione sepharose column and its activity was analyzed. The PHO85-PHO80 and PHO85-PAP1 kinase complexes were prepared via coimmunoprecipitation respectively. With purified recombinant PHO4 protein as substrate of the kinase complexes when the purified GST-ANK was added the kinase activity of PHO85-PHO80 or PHO85-PAP1 kinase complex was inhibited. These studies indicate that the ankyrin repeats interact with PHO85-PHO80 or PHO85-PAP1 complex inhibit their kinase activities and are crucial in interaction between PHO81 and other proteins.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 1999
PMID:Expression and Functional Analysis of Yeast PHO81 Ankyrin Repeats. 1213 13

PHO4 and PHO2 protein were overexpressed in E. coli and purified respectively. The gel retardation assays showed that PHO4 and PHO2 protein could bind to -401 -289 bp sequence of PHO81 gene promoter respectively. The assays of the mutant PHO2 proteins where the Ser-230 was changed into Asp (GST-2SD) or Ala (GST-2SA) showed the binding patterns were the same as the intact PHO2 protein. Footprinting results located the PHO4 binding site at the 354 -333 bp sequence of PHO81 gene PHO2 binding site at the -341 -334 bp. Therefore the region (-401 -289 bp) contains the upstream activation sequence (UAS) of PHO81 gene and PHO4 protein cooperates with PHO2 in acting on the upstream sequence of PHO81 gene and both of them regulate the expression of PHO81 gene.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 1999
PMID:PHO4 and PHO2 Protein Interact with Upstream Sequence of PHO81 Gene. 1213 14


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