Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Many complement inhibitors found in plants and other organisms have been recognized as an antiinflammatory drug. Sh-CRIT-ed1 is a complement inhibitory peptide, present on the Schistosoma parasite surface. In the present study, we expressed chemically synthesized oligonucleotides encoding Sh-CRIT-ed1 with an additional hexahistidine codon at the C-terminus and purified in Escherichia coli BL21. The cloned gene, which was multimerized four times in pBlue-script II KS (+) at the isoschizomer sites (BamHI, BglII), was named Sh4, and expressed in E. coli BL21 harboring pGEX-KG. The fusion protein (GST-Sh4) was purified with high yield successively by affinity chromatographies of glutathione-Sepharose 4B and Ni-NTA-agarose. Recombinant Sh-CRIT-ed1 was obtained readily by thrombin digestion and CNBr cleavage of GST-Sh4, and the yield was 9.03 mg from 1-liter culture of E. coli BL21 harboring pGEX-Sh4. The recombinant Sh-CRIT-ed1 showed strong anticomplementary activity (IC(50) = 6.02 microM) by complement haemolysis assay.
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PMID:Expression and purification of the anticomplementary peptide Sh-CRIT-ed1 (formerly Sh-TOR-ed1) as a tetramultimer in Escherichia coli. 1259 78

This work characterized the putative quinone oxidoreductase gene (qorA) from Staphylococcus aureus. The deduced amino acid sequence indicated that the 333 aa protein contains an NAD(P)H-binding motif. A Northern blot analysis revealed that 2.6 kb and 1.4 kb signals were detected by using a qorA probe. Both the signals were enhanced under the presence of a redox-cycling agent, 9,10-phenanthrenequinone (PQ). It was also revealed that the expression of three genes, SA1988, SA1989 (qorA) and SA1990, was enhanced at the transcriptional level by PQ exposure. The results suggested that the 2.6 kb signal detected by the qorA probe was in two co-transcripts, i.e. SA1990-qorA and qorA-SA1988 were transcribed. Besides, primer extension analyses confirmed the enhancement of qorA and SA1990 transcripts. The GST (glutathione S-transferase)-tagged QorA protein was expressed in Escherichia coli and purified using a glutathione affinity column. In purification steps, a 36 kDa band co-purified with the GST-QorA, and it was detected even in the thrombin-cleaved fraction. N-terminal amino acid sequences for the 36 kDa protein revealed that it was an intact QorA. They showed that QorA formed a multimer under physiological conditions. The purified recombinant GST-QorA catalysed NADPH consumption in the presence of PQ as a substrate, but not NADH. To characterize the catalytic activity of QorA, superoxide anion that was generated through one-electron reduction of PQ and hydroquinone that was produced by two-electron reduction of PQ were measured. During reduction of PQ by GST-QorA, superoxide anion was generated, whereas a small amount of 9,10-dihydroxyphenanthrene (hydroquinone of PQ) was produced. These results suggest that the activity of QorA is similar to zeta-Crystallin, catalysing an NADPH-dependent one-electron reduction of quinone.
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PMID:Oxidative-stress-inducible qorA encodes an NADPH-dependent quinone oxidoreductase catalysing a one-electron reduction in Staphylococcus aureus. 1262 1

In this study, we compared two gene fusion expression strategies using two rare codon genes (Ssh10b and MtGrxM) from archaea as a model system. Both genes can be highly expressed as N- or C-terminal fusion partners to GST or the intein/chitin-binding tag. However, the fusion protein with intein tag could not be cleaved, even under stringent conditions, possibly due to steric hindrance, thus preventing further purification. In contrast, the GST fusion system could increase protein expression level and the corresponding fusion protein could be easily cleaved by thrombin. After binding to glutathione sepharose, the fusion protein was cleaved on column, and a roughly purified protein fraction was eluted. This fraction was purified by heating at 80 degrees C for 10 min, followed by centrifugation. The correct total mass and N-terminal primary structure were confirmed by mass spectrometry and Edman degradation. Both constructs were used for in vitro expression, and similar results were obtained, indicating higher expression levels of the GST tag vs. intein/chitin tag. Taken together, our results suggest that the GST fusion system can be used as a considerable alternative to synthetic genes for the expression of rare codon genes. The affinity chromatography purification followed by a heating step is an efficient and convenient method for thermostable protein purification.
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PMID:High-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system. 1265 Oct 13

A major problem in assessing the vaccine and diagnostic potential of various proteins encoded by Mycobacterium tuberculosis genome is the inability to produce large quantities of these proteins, even when Escherichia coli or other heterologous systems are employed for recombinant protein production. To overcome these barriers, we have constructed a modified expression vector, using pGEX-4T-1 vector as the backbone. In addition to the features offered by the pGEX-4T vectors, the new vector allowed easy purification of recombinant proteins on the highly versatile Ni-NTA-agarose affinity matrix. The utility of the new vector was demonstrated by expressing and purifying, to near homogeneity, two M. tuberculosis proteins, i.e., Rv3872 (a member of the multi-gene PE subfamily) and Rv3873 (a member of the multi-gene PPE subfamily), which are encoded by the RD1 region of M. tuberculosis. The proteins encoded by rv3872 and rv3873 were expressed at high levels as fusion proteins with glutathione-S-transferase in E. coli. The recombinant Rv3872 and Rv3873 proteins were purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose and/or Ni-NTA-agarose affinity matrix and cleavage of the purified fusion proteins by thrombin protease. The recombinant Rv3872 protein was nearly homogeneous (more than 95% pure) while Rv3873 preparation was more than 90% pure. The recombinant Rv3872 and Rv3873 proteins were immunologically active and reacted with antibodies in sera from TB patients. Our results demonstrate the utility of the newly constructed expression vector with two affinity tags for efficient expression and purification of recombinant M. tuberculosis proteins expressed in E. coli, which could be used for further diagnostic and immunological studies.
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PMID:Construction of a modified vector for efficient purification of recombinant Mycobacterium tuberculosis proteins expressed in Escherichia coli. 1276 6

A cDNA encoding mouse implantation serine proteinase 2 ISP2 was amplified from total cDNAs of mouse uterus implantation sites on D4.5 of pregnancy by PCR, and sequenced GenBank accession No. A442918 . DNA sequencing indicated that the ISP2 cDNA had an unreported 204 bp sequence at 3' untranslated region besides the open reading frame encoding 279 amino acid residues, which was identical with literature. In order to obtain recombinant ISP2 rISP2 an expression plasmid pGEX-4T-2/ISP2 was constructed and transformed into E.coli BL21(DE3) strain. Expressed fusion protein GST-ISP2 was purified by SDS-PAGE and digested with thrombin, and the digestion mixture was subjected to SDS-PAGE again to recover rISP2. Rabbits were immunized using rISP2 as immunogen, and the polyclonal anti-ISP2 antisera were obtained. Immunohistochemical analysis using this antisera showed specific and high expression of ISP2 in mouse endometrial gland epithelium in early pregnancy.
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PMID:[Cloning, expression, and antibody production of mouse ISP2]. 1288 36

We previously localized the heparin binding region on high molecular weight kininogen to domain 5 (D5) by quantifying the binding using surface plasmon resonance of D5 fused at its N-terminal to glutathione-S-transferase. We further examined GST-(H475-S626) which at 100 nm was previously shown to be ineffective in reversing the heparin acceleration of antithrombin inhibition of thrombin. However, we now show that at a concentration of 400 nm, complete reversal of accelerated inhibition occurred. To characterize the interacting sequences on D5, four peptides representing surface loops of a molecular model were synthesized. Peptides H475-H485 and G440-G455, rich in histidine and low in lysine, showed weak or no detectable binding in the absence of Zn++, but tighter binding in the presence of Zn++. H483-K497 containing three histidines and six lysines showed tight binding without Zn++, and increased in avidity with Zn++. In contrast, G486-K502, low in histidine and high in lysine, showed tight binding (KD = 0.8 microm) in the absence and presence of Zn++. Both H483-K497 and G486-K502 were effective in neutralizing the accelerated inhibition by heparin of thrombin by antithrombin in the absence of Zn++. Therefore, a set of lysine residues in the sequence of K487-K502 is responsible for Zn++-independent binding of heparin. Further, a group of histidine residues in sequence range of H475-H485 contributes to Zn++-dependent binding of heparin to HK-D5.
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PMID:Fine mapping of the sequences in domain 5 of high molecular weight kininogen (HK) interacting with heparin and zinc. 1291 95

Photoreceptor rod cells contain a unique tetraspanin fusion protein known as peripherin/rds. This protein is important in membrane fusion events hypothesized to be essential to disk membrane morphogenesis and disk shedding. In vivo and in vitro fusogenic activity has been mapped to the C-terminal domain of peripherin/rds. Moreover, a fusion peptide domain localized to a 15 amino acid long region (residues 311-325) is essential for mediating lipid bilayer fusion of model membranes. To address the functional and structural properties required for peripherin/rds dependent membrane fusion, constructs of the entire C-terminal domain (residues 284-346) were generated and polypeptides expressed. A wild type-peripherin/rds C-terminal GST fusion construct that included the entire C-terminus (PERCTER) or a C-terminal truncation mutant (PERCTN) were engineered with a thrombin cleavage site. Protein expression was induced in E. coli with IPTG, expressed proteins cleaved from the GST with thrombin and purified to homogeneity on a Superdex 75 column. Purity was confirmed by SDS-PAGE and Western blot analysis. The purified wt C-terminal protein resolved as a monomer under reducing conditions on SDS-PAGE (15%) and was immunoreactive with anti peripherin/rds antibody 2B6 (gift from Dr R. Molday). The purified polypeptide promoted the requisite steps of fusion, membrane destabilization, lipid mixing and aqueous contents mixing. Conversely, the truncation mutant lacking a portion of the fusion domain was unable to promote these steps. A common feature of most membrane fusion proteins is a change in conformation upon membrane association. Structural changes in the C-terminal polypeptide were investigated using far UV CD. The far UV CD spectra of the purified C-terminal polypeptide indicated substantial alpha-helical content in the wt peptide in isotonic aqueous buffer. An increase in intensity of 208 and 222 nm CD bands upon addition of DPC vesicles indicated an increase in alpha-helical content of the polypeptide. These results demonstrate that a purified soluble form of the C-terminus of peripherin/rds can interact with biological phospholipids; moreover, this interaction promotes a conformational change that is most consistent with an increase in alpha-helical content.
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PMID:A soluble peripherin/Rds C-terminal polypeptide promotes membrane fusion and changes conformation upon membrane association. 1295 49

Receptor for activated C-kinase (RACK1) binds to protein kinase C and functions as an anchor for several other cellular components. Most in vitro studies of RACK1 have been carried out with RACK1 fused to a soluble fusion protein partner, such as GST or MBP. Here, we show that fusion complexes may exist as large soluble aggregates and thereby lead to false conclusions about the biological activity of RACK1. We developed a purification procedure that gave soluble monodisperse molecules of the protein. The RACK1 gene was cloned and expressed in a pMAL vector. After purification of the resulting MBP-RACK1 fusion protein, RACK1 was excised from MBP by thrombin, rendering RACK1 in a soluble monodisperse form as monitored by fluorimetric static light scattering, gel filtration, and ultracentrifugation. Circular dichroism analysis revealed that RACK1 was properly folded with a T(m) of approximately 62 degrees C and contained the predicted portions of secondary structures. The biological activity of the purified protein was verified by binding to activated protein kinase C. The production of soluble, high-purity RACK1 will allow structural studies and functional in vitro studies to identify interacting partners to this important scaffold protein.
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PMID:Expression and purification of receptor for activated C-kinase 1 (RACK1). 1296 40

The leukocyte beta2 integrin CR3 (CD11/CD18), is a surface heterodimeric glycoprotein that functions as a divalent cation-dependent adhesive complex. It mediates several important cell-substrate and cell-cell adhesive interactions among which the interaction with vascular endothelial cells that lead to leukocyte transmigration. We have isolated cDNA clones-coding for the rat complement receptor type 3 (CR3) alphaM subunit (CD11b) from a cDNA library. The cDNA sequence showed respectively 89.4% and 74.6% homology with its mouse and human counterpart. We have expressed the sequence coding for the VA module or Von Willebrand type domain (A-domain) and produced it in E. coli as a soluble recombinant fusion protein with GST. Simultaneously, we have cloned DNA fragments specific to the rat ICAM-1 domain 1 and domain 3 and expressed each clone in E. coli as recombinant soluble (rs) fusion proteins with GST. Recombinant CD11b A-domain was released from the fusion protein by thrombin cut. Purified ICAM-1 fusion peptides and CD11b A-domain were used to develop a direct binding assay that showed a specific binding between the rat ICAM-1 Ig like domain 3 and CD11b A-domain. These data demonstrate that the IgSF modules can be produced as a soluble recombinant fusion protein and used to study direct binding to the VA module displayed by members of the integrin superfamily.
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PMID:Cloning of the rat CR3 alphaM (CD11b) subunit, expression and binding assay of recombinant isolated CD11b VA (A-domain) and ICAM-1 Ig modules. 1507 40

In order to examine the elusive functional mechanism of GIF (Neuronal growth inhibitory factor, GIF) and elucidate the possible relationship between GIF and Alzheimer's disease, we constructed bait1 plasmid (pHyblex-GIF) by cloning GIF cDNA directly in frame with plasmid pHyblex, and used the yeast two-hybrid system to screen Alzheimer's disease human brain cDNA library and found the GIF-interacting proteins. The final results from coimmunoprecipitation and western blotting experiments confirmed that interacting proteins specifically binds to GIF. After sequencing the nucleotide of the putative positive plasmids and searching for homologues, we found that one of these is the part of human nuclear dUTPase protein sequence. Then the dUTPase genes are cloned into pGEX-4T-1, the fusion expression vector of GST,and highly expressed in E. coli BL21. The proteins dUTPase and GIF were purified and obtained by affinity chromatography, thrombin digestion and gel filtration on Sephacryl S100. It demonstrated that the proteins dUTPase and GIF had the growth inhibitory activity on co-cultured neuron in vitro. The inhibitory curve was very similar to the GIF. It's possible that dUTPase is one of the proteins interacting with GIF in Alzheimer's disease human brain extracts.
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PMID:[Molecular cloning and identification and biological activity of GIF-interacting protein(s)]. 1546 12


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