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)

We report the construction of an inducible, high-copy plasmid for the expression of foreign proteins in Escherichia coli. This plasmid, pPB1, combines the trc promoter, beta-galactosidase translation start site, and polylinker of pKK233-2 with the origin of replication region of pUC19. Replacement of the origin of replication of pKK233-2 results in a threefold increase in plasmid copy number of pPB1 compared with pKK233-2. Subclones of the cDNA for rabbit muscle fructose-1,6-bisphosphate aldolase (E.C. 4.1.2.13) in the two expression plasmids exhibit a comparable difference in copy number. An increase in protein expression measured by SDS-PAGE and aldolase specific activities reflects the increased copy number. Specific activities of aldolases in bacterial extracts differ approximately sixfold between the two expression plasmids in E. coli JM83. Aldolase A can compose up to 40% of the total protein in E. coli JM83 when expressed in pPB1, from which more than 100 mg of purified enzyme can be obtained per liter culture.
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PMID:Construction of a high-copy "ATG vector" for expression in Escherichia coli. 142 27

A rat oestrogen receptor-beta-galactosidase fusion protein was expressed using a pEX2/rat oestrogen receptor cDNA construct. Scatchard analysis of [3H]oestradiol-17 beta binding to the cell lysate revealed that the fusion protein had functional binding sites specific for oestradiol with a dissociation constant of 1.49 nmol/l. The relative molecular weight (M(r)) of the fusion protein was determined as 180,000 by immunoblot analysis of the cell lysate employing a monoclonal antibody to the human oestrogen receptor. The protein was isolated by means of SDS-PAGE and subsequent electroblotting. By immunization with the purified materials on nitrocellulose membrane, a polyclonal antibody to the rat oestrogen receptor was raised in a rabbit. Binding of [3H]oestradiol to the oestrogen receptor from the rat uterus was inhibited by the antibody in a dose-dependent manner. The antibody was also able to recognize the oestrogen receptor occupied by [3H]oestradiol. Thus, the antibody could react with both forms of the receptor molecule, either occupied or unoccupied by the hormone. In immunoblot analysis of the cytosol fraction of the rat uterus, a single band of M(r) 67,000, the size of the oestrogen receptor, was detected by the antibody. Moreover, when the antibody was applied to immunohistochemical examination of paraffin-embedded pituitary and brain sections of the rat, immunostaining was observed in cells of the anterior pituitary and in neurones in specific regions of the brain. The immunoreactivity was restricted exclusively to cell nuclei in both tissues.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A polyclonal antibody to the rat oestrogen receptor expressed in Escherichia coli: characterization and application to immunohistochemistry. 147 41

60% of the peripheral membrane skeleton of Euglena gracilis consists of equimolar amounts of two proteins (articulins) with M(r)s in SDS gels of 80 and 86 kD. To understand eventually how these proteins assemble and function in maintaining cell form and membrane integrity we have undertaken a molecular characterization of articulins. A lambda gt11 expression library constructed from Euglena gracilis mRNAs was screened with antibodies against both articulins. Two sets of cDNAs were recovered, and evidence from three independent assays confirmed that both sets encoded articulins: (a) Anti-articulin antibodies recognized a high molecular weight beta-galactosidase (beta-gal) fusion protein expressed in bacteria infected with lambda gt11 cDNA clones. (b) Antibodies generated against the bacterially expressed beta-gal fusion protein identified one or the other articulin in Western blots of Euglena proteins. These antibodies also localized to the membrane skeletal region in thin sections of Euglena. (c) Peptide maps of the beta-gal fusion protein were similar to peptide maps of Euglena articulins. From the nucleotide sequence of the two sets of cDNAs an open reading frame for each articulin was deduced. In addition to 37% amino acid identity and overall structural similarity, both articulins exhibited a long core domain consisting of over 30 12-amino acid repeats with the consensus VPVPV--V--. Homology plots comparing the same or different articulins revealed larger, less regular repeats in the core domain that coincided with predicted turns in extended beta-sheets. Outside the core domain a short hydrophobic region containing four seven-amino acid repeats (consensus: APVTYGA) was identified near the carboxy terminus of the 80-kD articulin, but near the amino terminus of the 86-kD articulin. No extensive sequence similarities were found between articulins and other protein sequences in various databanks. We conclude that the two articulins are related members of a new class of membrane cytoskeletal proteins.
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PMID:The two major membrane skeletal proteins (articulins) of Euglena gracilis define a novel class of cytoskeletal proteins. 152 17

A bacterial lacZ gene was inserted into an isolate of the Lymantria dispar nuclear polyhedrosis virus (LdMNPV). The transfer vector was constructed by site-directed mutagenesis of the translation start site of the LdMNPV polyhedrin gene, within the BglII E fragment of the viral genome. A multiple cloning sequence was inserted at this start site and used for the insertion of the lacZ gene into the transfer plasmid. Liposome transfection was used to cotransfect L. dispar tissue culture cells with viral DNA and the transfer plasmid. Recombinant LdMNPV isolates were purified by isolation of plaques producing beta-galactosidase but not polyhedra. Restriction enzyme fragment profiles were used to determine the site of the lacZ gene insertion, and DNA sequencing of the 5' and 3' ends of the lacZ gene insert and the adjoining polyhedrin promoter and coding regions was performed to identify its precise location. Expression of the lacZ gene was examined by studying virus-induced protein using [35S]methionine pulse-labelling, SDS-PAGE fractionation and autoradiography. Expression of beta-galactosidase was examined in tissue culture cells using colorimetric assays. The maximum rate of beta-galactosidase production was approximately 50 international units (IU)/10(6) tissue culture cells/day between 3 and 4 days post-infection (p.i), and the peak total expression was 158 IU/10(6) cells 5 days p.i. beta-Galactosidase activity was first detected 48 h p.i. in haemolymph samples from fourth instar L. dispar larvae injected with 10(6) p.f.u. of virus. The peak beta-galactosidase activity in larval haemolymph samples was 1931 IU/ml of haemolymph at 11 days p.i., just prior to death.
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PMID:Genetic engineering of a Lymantria dispar nuclear polyhedrosis virus for expression of foreign genes. 160 70

The capsid proteins of hepatitis A virus (HAV) were expressed as fusion proteins of beta-galactosidase in E. coli using the expression vector lambda gt11. Four fusion proteins were stably expressed and used to immunize rabbits to obtain mono-specific antisera. The antisera were unable to neutralize viral infectivity or react with HAV by radioimmunoassay. Three of the antisera were able to recognize HAV antigens in infected BS-C-1 cells by immunofluorescence and denatured capsid proteins by immunoblot analysis. The antisera were used to investigate the migration of the capsid proteins in gels by immunoblot analysis using standard SDS-PAGE conditions and in gels containing urea. The migration of VP1 and VP3 correlated with their molecular weights predicted from the nucleotide sequence and was consistent in either the presence or absence of urea. However, VP2 migrated with an apparent molecular weight significantly higher than the predicted value and, in gels containing urea, migrated as a doublet. It is proposed that the upper band of this doublet represents VP0, the proteolytic precursor of VP2 and VP4. The relative molecular mass (Mr) of VP4 was estimated to be less than 1 kDa, which is substantially lower than the 2.5 kDa predicted from the nucleotide sequence.
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PMID:Characterization of hepatitis A virus capsid proteins with antisera raised to recombinant antigens. 165 50

A human hippocampus cDNA library in lambda ZAP II was screened by hybridization with a rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase cDNA. Two clones (hh6 and hh3) were isolated and sequenced. The insert of clone hh6 was shown to correspond to the 3' end of the coding sequence of 50,000-Mr InsP3 3-kinase (referred to as 3-kinase-A). Sequencing of the clone hh3 insert yielded an open reading frame encoding a 472-amino acid protein with a calculated Mr of 53,451 (referred to as 3-kinase-B). The C-terminal part of 3-kinase-B (residues 187-462) was 68% identical with 3-kinase-A in amino acid sequence. The cDNA of clone hh3 was rescued as a Bluescript plasmid and expressed in Escherichia coli as a beta-galactosidase fusion product. It showed InsP3 3-kinase activity that was stimulated in the presence of Ca2+/calmodulin (more than 7-fold in a crude bacterial lysate from expressed plasmid). Regeneration of InsP3 3-kinase activity after SDS/PAGE identified a major polypeptide (Mr 60,000-65,000). The Km for InsP3 of expressed 3-kinase-B was 1.6 microM. These data provide molecular evidence for the existence of InsP3 3-kinase isoenzymes.
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PMID:Molecular cloning and expression of a new putative inositol 1,4,5-trisphosphate 3-kinase isoenzyme. 165 94

The 18 kDa and 32 kDa lectin binding proteins of Chlamydia trachomatis were characterized as glycoproteins by treatments with glycosidases. The proteins of the serovar L2 whole cell lysate were separated by SDS-PAGE and transferred to nitrocellulose paper. After treatment with an enzyme, the proteins were reacted with a biotinylated lectin. Each of the endoglycosidases tested affected the binding of the lectin to the protein. PNGase F inhibited the binding of Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), and Ulex europaeus agglutinin I (UEAI) to both the 18 kDa and 32 kDa proteins. Endoglycosidase F and H inhibited the binding of these lectins to the 32 kDa protein completely and to the 18 kDa protein partially. In the exoglycosidase treatments, alpha-L-fucosidase prevented binding of only UEAI to the two proteins while beta-galactosidase inhibited the binding of SBA. Mannosidase abolished the binding of all the lectins tested. Neuraminidase had no effect. The proteins isolated by electroelution from the excised gels after SDS-PAGE were digested with an endoglycosidase. PNGase F-treated proteins showed a lower molecular weight mobility in which the lectin binding ability was destroyed. Endo-alpha-N-acetylgalactosaminidase had no effect. The polysaccharide stain of isolated proteins with p-phenylenediamine showed a positive reaction. Radiolabeling with [3H]glucosamine did not reveal the 18 kDa and 32 kDa proteins in autoradiography but [3H]galactose did.
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PMID:The characterization of lectin-binding proteins of Chlamydia trachomatis as glycoproteins. 172 47

Avian beta 1,4 galactosyltransferase (GalTase) was purified from chicken serum, partially characterized and compared to mammalian GalTase using antibody cross-reactivity, Northern blot hybridization and amino acid sequence analysis. The enzyme was purified to apparent homogeneity by alpha-lactalbumin(LA)-agarose affinity chromatography followed by preparative SDS-polyacrylamide gel electrophoresis, and identified as two proteins of apparent molecular masses of 39 and 46 kD. Chicken serum GalTase had a Km for UDPGal of 42 microM, for GlcNAc of 10 mM and had optimal activity in the presence of 10-20 mM MnCl2. Substrate and linkage specificity analyses indicated that the purified enzyme behaves as a traditional Gal beta 1,4 GlcNAc:GalTase, since: (i) the avian beta 1,4 GalTase bound to alpha-LA; (ii) terminal GlcNAc residues served as good acceptors for chicken serum GalTase; (iii) the enzyme was inhibited by high concentrations of GlcNAc; (iv) the galactosylated product was sensitive to beta 1,4-specific beta-galactosidase. Finally, the disaccharide reaction product comigrated with authentic beta 1,4 N-acetyllactosamine standard. No other GalTase activities were detectable using a battery of defined glycoside substrates. Polyclonal antibodies raised against the two gel-purified GalTase proteins showed reactivity with avian GalTase by ELISA and immunoprecipitation assays. The antibodies also inhibited GalTase activity toward both high mol. wt and monosaccharide acceptor substrates. Despite similar kinetics and substrate specificity, the avian and mammalian GalTases showed little overall structural similarity, since polyclonal anti-avian GalTase IgG failed to react with mammalian GalTase purified from bovine milk, and conversely anti-bovine milk GalTase IgG did not react with the avian enzyme. Furthermore, in Northern blot analysis, no hybridization was detected when chicken embryo liver poly(A)+ RNA was probed with a mouse GalTase cDNA, even under conditions of reduced stringency. Amino acid sequence analysis identified three of five tryptic peptides that are homologous to the mammalian sequence within a putative substrate binding domain and the carboxy terminal domain of the enzyme. Their overall structural disparity leads us to believe that regions of homology between the avian and mammalian GalTases may represent active sites of the enzyme.
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PMID:Purification and characterization of avian beta 1,4 galactosyltransferase: comparison with the mammalian enzyme. 182 64

We have constructed a series of insertion mutations at 18 sites in the coding sequences of early region 1A (E1A) of human adenovirus type 5 (Ad5). At each site we have introduced three types of mutation: a 39-bp insertion specifying a 13-aa residue oligopeptide, a 39-bp insertion containing chain termination codons in all three reading frames, and a "collapsed" insert of 6-bp forming a conventional linker insertion mutation. All mutants were sequenced to determine the precise location, structure, and orientation of the inserts. The mutants were assayed for their abilities to trans-activate and to repress using transient expression assays in HeLa cells cotransfected with the E1A mutant plasmids and a reporter plasmid containing the bacterial beta-galactosidase (lac Z) gene under the control of Ad5 early promoters. The mutants were also tested for their ability to transform baby rat kidney cells in cooperation with either E1B or the ras oncogene. Each mutant was rescued into virus and infectivity was compared in HeLa and 293 cells. In addition, E1A protein synthesis was analyzed in cells infected with the mutant viruses and the insertions were found to have pronounced but unpredictable effects on electrophoretic mobility of E1A proteins in SDS-polyacrylamide gels. The results of functional assays indicated that only mutations mapping in, or deleting, the unique region of the 13 S mRNA product had any effect on ability to trans-activate and that a perfect correlation existed between ability of a mutant to trans-activate and to replicate efficiently in HeLa cells or to transform baby rat kidney cells in an E1A plus E1B mediated assay. In contrast, insertions near conserved region 2 of exon I and in the NH2-terminal portion of exon II significantly reduced repression activity but left transforming activity with E1B or with ras essentially unaffected suggesting that the repression function of E1A is separate from, or at least nonessential in, transformation.
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PMID:Isolation and characterization of insertion mutants in E1A of adenovirus type 5. 182 28

Developmental regulation of gamma-globin gene expression probably occurs through developmental-stage-specific trans-acting factors able to promote the interaction of enhancer elements located in the far upstream locus control region with regulatory elements in the gamma gene promoters and 3' A gamma enhancer located in close proximity to the genes. We have detected a nuclear protein in K562 and baboon fetal bone marrow nuclear extracts capable of binding to A+T-rich sequences in the locus control region, gamma gene promoter, and 3' A gamma enhancer. SDS/polyacrylamide gel analysis of the purified K562 binding activity revealed a single protein of 87 kDa. A K562 cDNA clone was isolated encoding a beta-galactosidase fusion protein with a DNA binding specificity identical to that of the K562/fetal bone marrow nuclear protein. The cDNA clone encodes a homeodomain homologous to the Drosophila antennapedia protein.
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PMID:A homeodomain protein binds to gamma-globin gene regulatory sequences. 187 Nov 39


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