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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Porcine type-I collagenase (Colg-1) was produced as a fusion protein in Escherichia coli using the pAX5 expression vector. The fusion protein consists of beta-galactosidase at the N terminus joined to a collagen hinge region and a blood-coagulation factor Xa cleavage site linked to Colg-1. Recombinant collagenase (reColg-1) was biologically active in the form of a fusion protein and could be released by treatment with factor Xa to yield Colg-1 with the authentic N terminus (phenylalanine) found in vivo. The results show that reColg-1 produced in E. coli is folded correctly, cleaves type-I collagen into 1/4 and 3/4 fragments at the characteristic Colg-sensitive site, and is produced at high enough levels to generate a source of recombinant enzyme for x-ray crystallography studies.
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PMID:Production in Escherichia coli of porcine type-I collagenase as a fusion protein with beta-galactosidase. 131 1

The intrinsic pathway of coagulation is initiated when zymogen factor VII binds to its cell surface receptor tissue factor to form a catalytic binary complex. Both the activation of factor VIIa and the expression of serine protease activity of factor VIIa are dependent on factor VII binding to tissue factor lipoprotein. To better understand the molecular basis of these rate-limiting events, the interaction of zymogen factor VII and tissue factor was investigated using as probes both a murine monoclonal antibody and a monospecific rabbit antiserum to human factor VII. To measure factor VIIa functional activity, a two-stage chromogenic assay was used; an assay which measures the factor Xa generated by the activation of factor VII to factor VIIa. Purified immunoglobulin from murine monoclonal antibody 231-7, which was shown to be reactive with amino acid residues 51-88 of the first epidermal growth factor-like (EGF) domain of human factor VII, inhibited the activation of factor VII to factor VIIa in a dose-dependent manner. The mechanism of this inhibition was demonstrated using a novel solid-phase ELISA which quantitatively measured the binding of purified factor VII zymogen to tissue factor adsorbed onto microtiter wells. Thus, the binding of factor VII zymogen to immobilized tissue factor was inhibited by antibody 231-7, again in a dose-dependent manner. Similar results were obtained using a monospecific rabbit antiserum to human factor VII which also reacted with the beta-galactosidase fusion proteins containing amino acid residues 51-88 (exon 4) of human factor VII. We conclude therefore that the exon 4-encoded amino acids of the first EGF domain of human factor VII constitute an essential domain participating in the binding of factor VII to tissue factor.
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PMID:The first epidermal growth factor domain of human coagulation factor VII is essential for binding with tissue factor. 137 73

A plasmid-encoding fusion protein interlinked by factor Xa recognition sequence between beta-galactosidase and a precursor of the small subunit of wheat ribulose-1,5-bisphosphate carboxylase has been constructed. The plasmid directed abundant synthesis of the fusion protein in Escherichia coli. The recombinant protein was accumulated in an aggregated form that was associated with the bacterial membranes. A procedure was developed to isolate the fusion protein in a relatively pure and soluble form. Bovine factor Xa cleaved the isolated chimera to generate the complete chloroplast precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase from the fused beta-galactosidase. The cleaved precursor protein was imported into the isolated chloroplasts and processed to yield its mature counterpart.
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PMID:An import-competent precursor of small subunit of ribulose-1,5-bisphosphate carboxylase generated by factor Xa cleavage from a beta-galactosidase fusion expressed in Escherichia coli. 139 14

A simple method is described to generate carrier-free recombinant antigens following their expression in Escherichia coli. A plasmid, called pMSgt11, has been constructed such that the cleavage site for the protease factor Xa separates the recombinant antigen from an enzymatically active beta-galactosidase. Thus, rapid purification of the active beta-galactosidase recombinant protein, followed by digestion with factor Xa, releases the antigen of interest. The pMSgt11 plasmid is compatible with the phage expression vector, lambda gt11 and the feasibility of applying this system has been demonstrated using malarial recombinant antigens. Inserts from lambda gt11 recombinant Plasmodium falciparum clones have been recloned into the EcoRI site of pMSgt11 and the expressed soluble fusion proteins have been purified from crude extracts using a one step affinity chromatography. After protease digestion, the fusion protein cleavage products were analysed by immunoblot with a panel of different human immune sera. We were able to successfully demonstrate specific antibody titers to the parasite-derived carrier-free antigen, without interference from anti-Escherichia coli-specific antibodies. The general application of this approach to epidemiological analysis is discussed.
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PMID:Parasite antigens expressed in Escherichia coli. A refined approach for epidemiological analysis. 210 15

The rev (art/trs) protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 K apparent molecular weight, is essential to target the mRNA for virion polypeptides into the cytoplasm. The rev protein was expressed in Escherichia coli as a beta-galactosidase fusion protein with a cleavage site for proteinase factor Xa. The rev-specific fragment was isolated to immunize mice. Five stable hybridoma cell lines were obtained producing monoclonal antibodies that reacted with rev protein in Western blot and ELISA. Using the monoclonal antibodies in indirect immunofluorescence, the rev protein could be localized in the nucleus, mostly in the nucleoli, of Hela cells that were transfected with a eukaryotic rev expression plasmid.
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PMID:Monoclonal antibodies directed against the rev protein of human immunodeficiency virus type 1. 217 12

A family of plasmid cloning vectors have been constructed, allowing both the sequencing and mutagenesis of foreign genes and the easy isolation of their expression products via fusion proteins in Escherichia coli. Fusion proteins can be inducibly expressed and isolated by affinity chromatography on APTG-Sepharose. The fusion protein consists of beta-galactosidase at the N-terminus, linked by a collagen 'hinge' region containing blood coagulation factor Xa cleavage site to the foreign protein at the C terminus. The factor Xa cleavage site at the N-terminal side of the foreign protein allows the release of the desired amino acid sequence under mild conditions. A multiple cloning site in all three reading frames and stop codons followed by the strong lambda t0 terminator facilitate simple gene insertions and manipulations. The intergenic region of the phage f1 inserted in both orientations allows the isolation of single-stranded DNA from either plasmid-strand for sequencing and mutagenesis. This vector family has been successfully used for the expression and purification of the isoleucyl-tRNA synthetase from Saccharomyces cerevisiae and the histidyl-tRNA synthetase from E. coli.
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PMID:The pAX plasmids: new gene-fusion vectors for sequencing, mutagenesis and expression of proteins in Escherichia coli. 222 22

By integrating fragments from the expression plasmids pJK2 and pJK4 into a derivative of the bacteriophage lambda, we constructed the phage expression vectors lambda JK2 and lambda JK4, which allow efficient cloning of genomic or cDNA either into the 5' end or the 3' end of the lacZ gene of Escherichia coli. Expression of barrier-free DNA in phase may lead to fusion proteins consisting of active beta-galactosidase (beta Gal) plus an additional polypeptide encoded by the inserted DNA. Analysis of distinct recombinant clones is quick and easy, due to the reversible integration of the plasmid into the genome. As an example, we constructed an expression library of genomic Plasmodium falciparum DNA in lambda JK2. We polymerised (amplified) and expressed a synthetic DNA fragment, which codes for a potential antigenic determinant of the 11-1 gene of Plasmodium falciparum as a fusion to the N terminus of active beta Gal. We demonstrate that such chimeric molecules can be affinity-purified and that polypeptides can be separated from the beta Gal part by cleavage with the protease factor Xa.
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PMID:A versatile phage lambda expression vector system for cloning in Escherichia coli. 246 28

The proteins p15 and p24 of the human immunodeficiency virus (HIV) type 1 gag gene were expressed as fusion proteins in Escherichia coli for detecting antibodies against the acquired immunodeficiency virus by Western blot (immunoblot) analysis. These fusion proteins contain amino acids 1 to 375 of the E. coli beta-galactosidase linked to the viral protein(s) by a recognition sequence for the specific protease factor Xa. They are obtained in large amounts in insoluble inclusion bodies. To avoid ambiguous results caused by cross-reaction of sera with bacterial proteins in Western blots, we purified the recombinant fusion proteins and subsequently removed the bacterial part of the fusions by cleavage with factor Xa. The cleavage mixtures were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The viral proteins obtained by this method did not contain any bacterial proteins or protein fragments. Thus, false-positive results in HIV Western blot analysis with bacterially expressed HIV proteins can be excluded with these purified recombinant viral antigens.
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PMID:Cleavage of procaryotically expressed human immunodeficiency virus fusion proteins by factor Xa and application in western blot (immunoblot) assays. 250 57

A semisynthetic winter flounder antifreeze proprotein (proAFP) coding region was constructed and inserted into a lacZ expression vector. ProAFP was produced from the vector in Escherichia coli as a C-terminal fusion to the first 289 amino acids of beta-galactosidase (beta-gal). The proAFP and beta-gal domains of the beta-gal-proAFP fusion protein were separated by the recognition signal for the blood coagulation protease, factor Xa. Upon induction with isopropylthio-beta-D-galactoside the fusion protein accumulated to levels of 15% of the total protein. The beta-gal-proAFP fusion protein was partially purified by differential centrifugation, but required solubilization prior to factor Xa digestion. The solubilized fusion protein was efficiently and correctly cleaved by factor Xa, after which the proAFP was purified by gel permeation. Bacterial proAFP was indistinguishable from natural proAFP by the criteria of antifreeze activity, amino-terminal sequence (15 cycles), reverse-phase HPLC and SDS-polyacrylamide gel electrophoresis. Circular dichroism measurements showed that proAFP is a composite of random coil and alpha-helical secondary structure, with an alpha-helix content of 44% at 0 degrees C. It seems probable that the C-terminal region of proAFP, which corresponds to the mature AFP protein, is mainly alpha-helical, and that the N-terminal pro-segment is random coiled.
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PMID:Biosynthesis of winter flounder antifreeze proprotein in E.coli. 268 48

A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli. The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin. The fusion protein contains the recognition sequence (Ile-Glu-Gly-Arg) for blood coagulation factor Xa protease between the two domains. Cleavage by factor Xa separates the two domains and the target protein domain can then be purified away from the MBP domain by repeating the affinity chromatography step. A prokaryotic (beta-galactosidase) and a eukaryotic (paramyosin) protein have been successfully purified by this method.
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PMID:An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. 307 5


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