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)

Synthetic genes for human insulin A and B chains were cloned separately in plasmid pBR322. The cloned synthetic genes were then fused to an Escherichia coli beta-galactosidase gene to provide efficient transcription and translation and a stable precursor protein. The insulin peptides were cleaved from beta-galactosidase, detected by radioimmunoassay, and purified. Complete purification of the A chain and partial purification of the B chain were achieved. These products were mixed, reduced, and reoxidized. The presence of insulin was detected by radioimmunoassay.
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PMID:Expression in Escherichia coli of chemically synthesized genes for human insulin. 8

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

The glycoprotein complex gp58/116 of human cytomegalovirus (HCMV) represents a dominant antigen for the humoral immune response. We have used the human monoclonal antibody C23, which is capable of neutralizing HCMV in tissue culture without the addition of complement, to study the origin of gp116 as well as the amino acid sequence recognized by the antibody. Our results show that gp116 is derived from the same open reading frame as gp58 and that it represents the amino-terminal portion of the precursor protein. Using prokaryote-expressed beta-galactosidase-gp116 fusion proteins, the binding site of C23 was located to between amino acids 27 to 84 of the amino-terminal portion of gp116. Analyses of HCMV-positive human sera revealed that this portion of the molecule is immunogenic during natural infection.
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PMID:The gp116 of the gp58/116 complex of human cytomegalovirus represents the amino-terminal part of the precursor molecule and contains a neutralizing epitope. 170 66

sigma E, a major sporulation-specific sigma factor of Bacillus subtilis, is derived from an inactive precursor protein (pro-sigma E). The formation of sigma E from pro-sigma E requires the products of several stage II genes, including spoIIGA, a gene that is cotranscribed with the pro-sigma E coding region (spoIIGB, or sigE). SpoIIGA has been hypothesized to be both a membrane-bound protein and the protease which converts pro-sigma E into sigma E. to learn more of its properties, we joined the Escherichia coli lacZ gene to the 3' end of spoIIGA as a translational fusion, creating a gene whose product was found to contain both beta-galactosidase and SpoIIGA activities. Assaying for the beta-galactosidase activity of the chimeric protein as a measure of its abundance, we determined that the spoIIGA::lacZ product accumulated to approximately 10% the level of a spoIIGB::lacZ fusion protein. Using differential centrifugation to fractionate B. subtilis extracts that contained beta-galactosidase fusion proteins, we observed that the beta-galactosidase activity of the spoIIGA::lacZ fusion protein was preferentially associated with a Triton X-100-sensitive, fast-sedimenting portion of the extract, while the beta-galactosidase activity of the spoIIGB::lacZ fusion protein remained primarily in the supernatant fraction. If the properties of the fusion proteins are assumed to be representative of those of the products of the genes to which lacZ is joined, these results support the hypothesis that SpoIIGA is a membrane-bound protein that acts catalytically in the processing of pro-sigma E into sigma E.
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PMID:Synthesis and fractionation properties of SpoIIGA, a protein essential for pro-sigma E processing in Bacillus subtilis. 174 37

Saposins are small, heat-stable glycoproteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. Saposins A, B, C, and D are derived by proteolytic processing from a single precursor protein named prosaposin. Saposin B, previously known as SAP-1 and sulfatide activator, stimulates the hydrolysis of a wide variety of substrates including cerebroside sulfate, GM1 ganglioside, and globotriaosylceramide by arylsulfatase A, acid beta-galactosidase, and alpha-galactosidase, respectively. Human saposin B deficiency, transmitted as an autosomal recessive trait, results in tissue accumulation of cerebroside sulfate and a clinical picture resembling metachromatic leukodystrophy (activator-deficient metachromatic leukodystrophy). We have examined transformed lymphoblasts from the initially reported saposin B-deficient patient and found normal amounts of saposins A, C, and D. After preparing first-strand cDNA from lymphoblast total RNA, we used the polymerase chain reaction to amplify the prosaposin cDNA. The patient's mRNA differed from the normal sequence by only one C----T transition in the 23rd codon of saposin B, resulting in a threonine to isoleucine amino acid substitution. An affected male sibling has the same mutation as the proband and their heterozygous mother carries both the normal and mutant sequences, providing additional evidence that this base change is the disease-causing mutation. This base change results in the replacement of a polar amino acid (threonine) with a nonpolar amino acid (isoleucine) and, more importantly, eliminates the glycosylation signal in this activator protein. One explanation for the deficiency of saposin B in this disease is that the mutation may increase the degradation of saposin B by exposing a potential proteolytic cleavage site (arginine) two amino acids to the amino-terminal side of the glycosylation site when the carbohydrate side chain is absent.
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PMID:Characterization of a mutation in a family with saposin B deficiency: a glycosylation site defect. 232 May 74

The molecular basis of egg formation in the parasitic liver fluke, Fasciola hepatica, was investigated by isolating and characterizing an abundant cDNA from a female genital complex cDNA library. It was expressed in Escherichia coli as a beta-galactosidase fusion protein, which was purified and used to produce polyclonal antibodies. Using immunoblots, the antiserum recognized two soluble constituents of isolated egg shells, both significantly larger than predicted from cDNA sequencing. Using in situ hybridization, the message was detected in cells in the adult vitelline follicles. Eggshell protein mRNA expressed in E. coli will provide a source of precursor protein for further studies of parasite eggshell formation.
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PMID:Identification, expression and in situ hybridization of an eggshell protein gene from Fasciola hepatica. 251 33

The complete mouse prepro-nerve growth factor (NGF) DNA was fused to the carboxyl terminus of the beta-galactosidase (lac-z) gene of Escherichia coli. Similarly, a genomic fragment encoding the human NGF comprising codons 11 to 106 (from a total of 118) was fused to the fifth codon of the amino terminus of beta-galactosidase. Both bacterial vectors produce high amounts of the chimeric proteins. After cell lysis most of the chimeric mouse preproNGF protein is insoluble and appears in the pellet, whereas the majority of the chimeric human beta-NGF remains in the supernatant. Purification of the fusion proteins from the soluble fraction was achieved by affinity chromatography to p-aminophenyl beta-D-thio-galactoside Sepharose. Yields of the purified chimeric proteins were increased threefold to fourfold by the addition of protease inhibitors in the lysis and chromatography buffers. Their antigenic similarity to the preproNGF and mouse beta-NGF was examined by their interaction to sera raised against synthetic peptides which reproduce sequences of the precursor protein and to sera directed against native and denatured mouse beta-NGF using enzyme-linked immunoabsorbent assay (ELISA) techniques. Antibodies to the peptide N2 (-163 to -139) interacted with high affinity with the chimeric mouse preproNGF protein. Antisera to native and denatured mouse beta-NGF interacted with both chimeric proteins but with a variable degree of affinity. These results provide direct evidence that certain antisera to mouse beta-NGF can cross-react with the human beta-NGF molecule.
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PMID:Synthesis of chimeric mouse nerve growth factor precursor and human beta-nerve growth factor in Escherichia coli: immunological properties. 264 14

A cDNA library was prepared in lambda gt11 bacteriophage from poly(A)+ RNA isolated from primary cultures of endothelial cells from human umbilical vein. Approximately 2.5 million independent recombinants were screened and 2 of those were found to synthesize a fusion protein with beta-galactosidase that reacted with rabbit antibody against human von Willebrand factor. Comparison of the amino acid sequence translated from the cDNA insert of the two clones with the amino acid sequence determined by Edman degradation of the protein established that both phage isolates code for von Willebrand factor. The first clone (lambda HvWF1) contained an insert of 404 nucleotides that corresponded to amino acid residues 1-110 in the mature protein circulating in blood, in addition to a portion (24 amino acids) of a prepro leader sequence. The second cDNA clone (lambda HvWF3) contained an insert of 4.9 kilobases that coded for the carboxyl-terminal 1525 amino acids of von Willebrand factor, a stop codon of TGA, 134 nucleotides of 3' noncoding sequence, and a poly(A) tail of 150 nucleotides. The two clones together code for greater than 80% of the molecule circulating in blood. The same carboxyl-terminal lysine residue was identified in the mature protein as well as in the cDNA, indicating that all of the proteolytic processing that occurs during the biosynthesis and assembly of von Willebrand factor is associated with the amino-terminal portion of the precursor protein. The amino acid sequence of von Willebrand factor indicates the presence of two different internal gene duplications and one triplication. These repetitive amino acid sequences account for about one-half of the amino acids present in the mature protein. The tetrapeptide sequence of Arg-Gly-Asp-Ser, which mediates the cell attachment and platelet binding activity of fibronectin, was also identified in the carboxyl-terminal portion of von Willebrand factor.
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PMID:Cloning and characterization of two cDNAs coding for human von Willebrand factor. 286 88

Hydrophobic surfactant-associated protein of Mr 6000-14,000 was isolated from ether/ethanol or chloroform/methanol extracts of mammalian pulmonary surfactant. Automated Edman degradation in a gas-phase sequencer showed the major N-terminus of the human low molecular weight protein to be Phe-Pro-Ile-Pro-Leu-Pro-Tyr-Cys-Trp-Leu-Cys-Arg-Ala-Leu-. Because of the N-terminal phenylalanine, the surfactant protein was designated SPL(Phe). Antiserum generated against hydrophobic surfactant protein(s) from bovine pulmonary surfactant recognized protein of Mr 6000-14,000 in immunoblot analysis and was used to screen a lambda gt11 expression library constructed from adult human lung poly(A)+ RNA. This resulted in identification of a 1.4-kilobase cDNA clone that was shown to encode the N-terminus of the surfactant polypeptide SPL(Phe) (Phe-Pro-Ile-Pro-Leu-Pro-) within an open reading frame for a larger protein. Expression of a fused beta-galactosidase-SPL(Phe) gene in Escherichia coli yielded an immunoreactive Mr 34,000 fusion peptide. Hybrid-arrested translation with this cDNA and immunoprecipitation of [35S]methionine-labeled in vitro translation products of human poly(A)+ RNA with a surfactant polyclonal antibody resulted in identification of a Mr 40,000 precursor protein. Blot hybridization analysis of electrophoretically fractionated RNA from human lung detected a 2.0-kilobase RNA that was more abundant in adult lung than in fetal lung. The larger RNA and translation product indicates that SPL(Phe) is derived by proteolysis of a large polypeptide precursor. The amino acid sequence of the predicted protein, beginning Phe-Pro-Ile-Pro-Leu-Pro-Try-, comprises a hydrophobic peptide that is a major protein component of surfactant lipid extracts used successfully to treat hyaline membrane disease in newborn infants. These proteins, and specifically SPL(Phe), may therefore be useful for synthesis of replacement surfactants for treatment of hyaline membrane disease in newborn infants or of other surfactant-deficient states.
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PMID:cDNA and deduced amino acid sequence of human pulmonary surfactant-associated proteolipid SPL(Phe). 303 61

Human lactase-phlorizin hydrolase [EC 3.2.1.23-3.2.1.62] is a disaccharidase located in the microvillus membrane of small intestinal epithelial cells. The enzyme is synthesized as a precursor protein in the endoplasmic reticulum and in addition to being glycosylated is subsequently proteolytically processed to the mature microvillus membrane-bound form after passing the trans-Golgi compartment. We studied the oligomerization of human lactase-phlorizin hydrolase in transfected polarized Madin Darby canine kidney cells using metabolic labeling and sucrose-density centrifugation analysis. We detected high mannose dimers of the lactase-phlorizin hydrolase precursor molecule after metabolic labeling with [35S]methionine at 37 and 15 degrees C. In addition, both complex-glycosylated lactase-phlorizin hydrolase precursor molecule and the mature microvillus membrane-bound enzyme showed this oligomeric structure. Chemical crosslinking resulted in the detection of covalently crosslinked lactase-phlorizin hydrolase dimers after sodium dodecyl sulfate polyacrylamide gel electrophoresis. These results provide evidence that oligomerization of lactase-phlorizin hydrolase is an early event and begins in the endoplasmic reticulum.
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PMID:Human lactase-phlorizin hydrolase: evidence of dimerization in the endoplasmic reticulum. 748


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