Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.6 (thromboplastin)
 13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasmid expression vectors have been constructed that direct the synthesis of foreign polypeptides in Escherichia coli as fusions with the C terminus of Sj26, a 26-kDa glutathione S-transferase (GST; EC 2.5.1.18) encoded by the parasitic helminth Schistosoma japonicum. In the majority of cases, fusion proteins are soluble in aqueous solutions and can be purified from crude bacterial lysates under non-denaturing conditions by affinity chromatography on immobilised glutathione. Using batch wash procedures several fusion proteins can be purified in parallel in under 2 h with yields of up to 15 micrograms protein/ml of culture. The vectors have been engineered so that the GST carrier can be cleaved from fusion proteins by digestion with site-specific proteases such as thrombin or blood coagulation factor Xa, following which, the carrier and any uncleaved fusion protein can be removed by absorption on glutathione-agarose. This system has been used successfully for the expression and purification of more than 30 different eukaryotic polypeptides.
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PMID:Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. 304 11

The alpha subunits of the leukocyte CD11/CD18 integrins contain an approximately 200 amino acid 'inserted' or I domain. The I domain of the cell-surface Mac-1 (CD11b/CD18) integrin has been shown to be the major recognition site for several adhesion ligands, including iC3b, fibrinogen, factor X, and ICAM-1. The I domain from the Mac-1 alpha subunit has been expressed in Escherichia coli as a soluble GST-fusion protein containing a factor Xa sensitive cleavage site. Analytical characterization of the purified I domain reveals that it is obtained in very high quality at high yields. CD and NMR spectra indicate that I domain adopts a predominantly folded structure in solution, independent of the remainder of the alpha subunit. Addition of Ca2+ and Mg2+ did not significantly perturb the structural conformation.
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PMID:Purification and structural characterization of the CD11b/CD18 integrin alpha subunit I domain reveals a folded conformation in solution. 764 57

A truncated variant of the hepatitis B virus X gene (HBx) was cloned into the fusion expression vector of pGEX-3X (Pharmacia), resulting in a GST-HBx fusion gene construction (pGEX-3XXBF). This plasmid was transformed into and expressed by the Escherichia coli strain DH5. More than 80% of the expressed fusion protein was found in the insoluble fraction (inclusion body) of the cell lysate. The fusion protein was selectively extracted from the inclusion bodies with 8 M urea at pH 6.5, and it was refolded by diluting 3-fold with deionized distilled water at 4 degrees C. The in vitro cleavage of the refolded fusion protein by factor Xa at about 2-3 mg ml-1 in the presence of 2.66 M urea at pH 6.5 was complete. The final steps of purification involved precipitation of the cleaved proteins with ammonium sulphate, solubilization in guanidine hydrochloride and separation on a Superdex 75 FPLC column. With this approach, following an inclusion body strategy and a beneficial in vitro refolding, a predominantly hydrophobic and highly disulphide-bonded protein was produced in preparative scale for subsequent diagnostic use.
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PMID:An alternative purification protocol for producing hepatitis B virus X antigen on a preparative scale in Escherichia coli. 930 71

The cDNA coding for the mature hypodermin C from first instars of Hypoderma lineatum was cloned by reverse transcription and PCR amplification of total larval RNA using specific oligonucleotide primers. This cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein. Mature hypodermin C was released from the GST-fusion after glutathione-Sepharose 4B affinity chromatography and proteolytic cleavage using factor Xa. The purified recombinant protein showed enzymatic activity in gelatin-polyacrylamide gels and when azocoll was used as substrate. An enzyme-linked immunosorbent assay was developed using the recombinant antigen. Positive/negative cutoff values were calculated using the mean OD percentage (1.74%) of 113 negative sera plus three standard deviations. Sensitivity and specificity according to the resulting cutoff (10.74%) were 85 and 98.2% respectively.
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PMID:Hypoderma lineatum: expression of enzymatically active hypodermin C in Escherichia coli and its use for the immunodiagnosis of hypodermosis. 970 25

The E6 Zn(2+)-binding protein of high-risk human papillomaviruses (HPVs) is one of the major transforming proteins encoded by these tumor viruses. A bacterial system was used to express wild type and truncated forms of HPV-16 E6 linked to GST. The recombinant proteins were released from GST through cleavage of a factor Xa site. Functional analysis of these proteins demonstrated that amino acids 2--142 comprise the minimal domain of E6 required to promote the degradation of p53 in vitro in a rabbit reticulocyte lysate. This purified protein, E6(Delta 143--151), required a high salt concentration for maximum solubility, eluted as a monomer on gel filtration, and was shown to bind two Zn(2+) ions by atomic absorption analysis. An N-terminal subdomain of E6 (amino acids 2--77, E6-N) was similarly purified. Unlike E6(Delta 143--151), E6--N was very soluble in low-salt buffers and hence was highly amenable to biophysical characterization. E6-N was shown to bind one Zn(2+) ion by electrospray mass spectrometry and by atomic absorption analysis. UV--visible spectroscopic analysis of Co(2+)-substituted E6--N revealed that four cysteine residues coordinate the metal ion. Mutational studies of all the cysteine residues in E6--N substantiated a critical role for Cys 30, 33, 63, and 66 in Zn(2+) binding and in proper folding of the subdomain. Equilibrium sedimentation of E6-N demonstrated that it is a monomer, like E6(Delta 143--151), at low concentrations, but dimerization occurs at high concentrations (K(d) = 0.1 mM). Finally, circular dichroism studies revealed significant secondary structure for both E6(Delta 143--151) and E6--N. The results support a model of monomeric E6 possessing two functionally critical Zn(2+)-binding motifs.
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PMID:Purification and biophysical characterization of a minimal functional domain and of an N-terminal Zn2+-binding fragment from the human papillomavirus type 16 E6 protein. 1117 Apr 44

Osteoprotegerin ligand (OPGL) is a key regulator of formation and activation of osteoclasts. In the present study, the cDNA encoding the extracellular domain of murine OPGL (sOPGL) was synthesized by RT-PCR and cloned into fusion expression vector pET-42a(+) in a certain strategy on purpose that the fusion tag could be completely removed by factor Xa from the expressed fusion protein without any vector-encoded sequence left. Induced with IPTG, the recombinant E. Coli cells produced a 47 kD protein in high level that could be recognized, through Western blotting analysis, by the antibody against OPGL. The expressed products were purified through Glutathione-sepharose 4B affinity chromatography. Along with the fusion molecule, a protein about 30 kD was also specifically bound to the resin. The 30 kD molecule could be recognized by polyclonal antibody against GST-IGF-1, but not by antibody against OPGL. It suggested that the 30 kD molecule was derived from the degradation of the fusion protein. After the cleavage with factor Xa and further purification, the fusion tag was removed and the recombinant sOPGL was obtained. Finally, we confirmed that the recombinant sOPGL could promote osteoclast formation from mouse bone marrow cells in a dose dependent manner.
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PMID:Expression, purification and bioactivity characterization of extracellular domain of murine osteoprotegerin ligand. 1547 18

A gene coding for phosphoketolase, a key enzyme of carbohydrate catabolism in heterofermentative lactic acid bacteria (LAB), was cloned from a Lactobacillus paraplantarum C7 and expressed in Escherichia coli. The gene is 2502 bp long and codes for a 788-amino-acids polypeptide with a molecular mass of 88.7 kDa. A Shine-Dalgamo sequence (aaggag) and an inverted-repeat terminator sequence are located upstream and downstream of the phosphoketolase gene, respectively. The gene exhibits an identity of >52% with phosphoketolases of other LAB. The phosphoketolase of Lb. paraplantarum C7 (LBPK) contains several highly conserved phosphoketolase signature regions and typical thiamine pyrophosphate (TPP) binding sites, as reported for other TPP-dependent enzymes. The phosphoketolase gene was fused to a glutathione S-transferase (GST::LBPK) gene for purification. The GST::LBPK fusion protein was detected in the soluble fraction of a recombinant Escherichia coli BL21. The GST::LBPK fusion protein was purified with a yield of 4.32 mg/400 ml by GSTrap HP affinity column chromatography and analyzed by N-terminal sequencing. LBPK was obtained by factor Xa treatment of fusion protein and the final yield was 3.78 mg/400 ml. LBPK was examined for its N-terminal sequence and phosphoketolase activity. The K(M) and Vmax values for fructose-6-phosphate were 5.08 +/- 0.057 mM (mean +/- SD) and 499.21 +/- 4.33 micromol/min/mg, respectively, and the optimum temperature and pH for the production of acetyl phosphate were 45 degrees C and 7.0, respectively.
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PMID:Cloning and characterization of a gene encoding phosphoketolase in a Lactobacillus paraplantarum isolated from Kimchi. 1805 5

Shiga toxin family comprises toxins belonging to two major groups, Stx1 and Stx2, produced by the bacteria Shigella dysenteriae and some strains of Escherichia coli. Shiga toxins are the leading cause of diarrhea associated with life threatening hemolytic uremic syndrome (HUS). StxA is a ribosome inactivating protein (RIP) which inhibits the protein synthesis in most species of prokaryotes and eukaryotes. An in vitro expression system has not been reported to produce full-length biological active StxA subunit; hence substantial progress has been hampered. In the present study, a DNA fragment (955 bp Gene Bank Accn No HM017965) encoding for subunit A of Stx was amplified from Shigella dysenteriae type 1 and subsequently cloned in pGEX-5X-2 vector. We successfully produced recombinant StxA as GST fusion protein in Escherichia coli using pGEX-5X-2-STXA construct under IPTG induction. For the purpose of immunization the GST-tag was removed by factor Xa mediated endoproteolytic cleavage from GST-StxA. Antisera raised against rStxA in mice reacted with recombinant purified protein of rStxA and lysate of Shiga toxin. It was shown that antisera produced against rStxA significantly recognized Stx producing strains of S. dysenteriae and E. coli. The antiserum produced effectively neutralized the Shiga toxin's cytotoxicity towards Vero cells.
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PMID:Expression, Purification and Immunological Characterization of Recombinant Shiga Toxin A Subunit. 2628 29