Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CEDIA assays represent a state of the art technique utilizing two genetically engineered, enzymatically inactive fragments of beta-galactosidase as the basis for a homogeneous enzyme immunoassay. The smaller, amino-terminal polypeptide, designated the enzyme donor (ED), can recombine spontaneously with the large residual fragment, called the enzyme acceptor (EA), to form active beta-galactosidase, in a process called complementation. ED have been designed in such a way that a ligand, such as a hormone or drug, can be chemically attached to a specific amino acid residue without affecting the enzyme complementation. However, the binding of a ligand-specific antibody to the ED-ligand conjugate will inhibit complementation. If a sample containing ligand is added to the reaction mixture, the ligand will compete with the ED-ligand conjugate for the limited number of antibody binding sites. Thus, the ligand concentration in the sample will modulate enzymatic activity by influencing the amount of free ED-ligand conjugate available for complementation. The basic technology of CEDIA assays has a number of inherent advantages, the most important of these being a linear calibration curve with high precision over the whole assay range, lack of endogeneous enzyme activity and minimal serum interference, chemically defined conjugates and flexibility in assay design. These provide significant advantages in comparison to other homogeneous immunoassay techniques. As a result, CEDIA assays have been successfully developed for high concentration drugs such as theophylline, phenobarbital and phenytoin as well as for very low concentration analytes such as digoxin, B12 and folate. In a modified assay format, even the determination of binding proteins has been accomplished, an example being thyroxine binding proteins in the CEDIA T-uptake assay. More recently, the methodology has been extended to the measurement of high molecular weight analytes like ferritin.
 ...
PMID:CEDIA in vitro diagnostics with a novel homogeneous immunoassay technique. Current status and future prospects. 161 62

Many virally encoded proteinases cleave themselves out of a polyprotein, with cleavage occurring usually at their own N terminus. This property was used to develop an in vivo screening system using the lacZ gene fragment of M13mp18. When a fusion protein of the alpha fragment of beta-galactosidase and an active 2A proteinase of human rhinovirus 2 was expressed, alpha complementation was not affected, as the 2A proteinase cleaved itself off the alpha fragment. However, fusion of an inactive 2A prevented alpha complementation, as the 2A polypeptide remained fused to the alpha fragment. After random mutation of the 2A gene by PCR amplification, mutants were screened; M13 phage defective in alpha complementation were obtained at an efficiency of 5% and were shown to contain mutated 2A genes. Intermolecular cleavage was then examined by expressing an alpha fragment-inactive proteinase fusion protein as substrate for an active 2A proteinase expressed from an M13 vector. alpha complementation indicated intermolecular processing of the 2A cleavage site on the alpha fragment-inactive proteinase fusion protein. This versatile system thus allows the high-density screening of both active and inactive proteinase mutants, cleaving either intramolecularly or intermolecularly, and should be applicable to other proteinases of high specificity.
 ...
PMID:Proteinase trapping: screening for viral proteinase mutants by alpha complementation. 164 26

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.
 ...
PMID:Molecular cloning and expression of a new putative inositol 1,4,5-trisphosphate 3-kinase isoenzyme. 165 94

Somatostatin gene fragment extracted and purified from plasmid pSom5 bacterium was ligated with the plasmid pBD2 DNA. Transformation of E. coli D29A1 with the chimeric plasmid DNA led to the synthesis of a polypeptide including the sequence of amino acids corresponding to somatostatin. The chimeric protein (50000 dalton) was purified and characterized by the beta-galactosidase affinity chromatography and the expression of the somatostatin gene in E. coli D29A1 is certain after the radioimmunoassay of the chimeric protein and its mixture by treatment with cyanogen bromide.
 ...
PMID:[Expression of somatostatin gene in E. coli D29A1]. 167 42

Examination of the hemagglutinating activity of extracts from seeds of Erythrina corallodendron at various maturation stages revealed that the level of lectin increases markedly past mid-maturation. Seeds at this stage of maturation served as a source of mRNA for the construction of an expression cDNA library in the vector lambda Zap, which generates fusion proteins with an N-terminal portion of beta-galactosidase. The library was screened with rabbit polyclonal anti-ECorL antiserum. Four immunopositive clones were isolated. Western blot analysis of cell extracts from one of the clones (pIEcL-B) showed a 36 kDa protein that reacted with the antiserum, as well as with a mouse monoclonal antibody raised against the lectin. DNA sequence analysis by the chain termination method revealed that clone pIEcl-C has an insert of 1017 bp with the entire coding sequence of ECorL, beginning with an initiation codon ATG at position 26 and ending with stop codon TAA at position 868. This fragment encodes a polypeptide of 281 amino acids consisting of a signal leader sequence of 25 amino acids and a mature protein of 256 amino acids. The deduced amino acid sequence from this fragment is identical to the sequence of the first 244 amino acids of ECorL, as determined at the protein level, except at 7 positions.
 ...
PMID:Cloning and sequence analysis of the Erythrina corallodendron lectin cDNA. 169 39

The gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from T. spiralis infective larvae was ligated into phage vector lambda gt11 DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gt11 expression library was constructed. A cDNA clone encoding a 46 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing nearly full-length cDNA for a 46 kDa protein was isolated. The gene encoding this 46 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 46 kDa polypeptide. The antigenic polypeptide was excreted/secreted as a 46 kDa native antigen. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.
 ...
PMID:Molecular analysis of the gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae. 169 76

Marek's disease virus (MDV) gene clones, RA2 and GA8, constructed in E. coli bacteriophage lambda-gt11 (gt11) were identified by a monoclonal antibody (MAb), H19.47, against a putative transformation-related viral antigen consisting of a complex of three phosphorylated polypeptides, pp41, pp38, and pp24. Both recombinants have a MDV-DNA insert of about 0.5 kb and are mapped to the region of BamHI-H or EcoRI-X fragments of the MDV genome by Southern blot hybridization. Immunoblot and immunoprecipitation with H19.47 identified a recombinant beta-galactosidase-MDV 140-kD fusion protein for RA2 and a 127-kD fusion protein for GA8. Immunoprecipitation of 35S-methionine-labeled, MDV-infected chicken embryo fibroblasts (CEF) with antisera against RA2 and GA8 fusion proteins recognized five polypeptides, of which three (p41, p38, and p24) are specified by H19.47 and the remaining two, p135 and p20, have not been previously identified. Immunoprecipitation of 32P-phosphate-labeled or 3H-glucosamine-labeled, GA-MDV-infected CEF with the antiserum against RA2 fusion protein identified a phosphorylated polypeptide of 38 kD and two glycoproteins of 60 and 49 kD, respectively. The antisera against recombinant fusion proteins thus revealed the existence of epitopes common to the phosphorylated polypeptides and other MDV-specific polypeptides. Sera from chickens or mice hyperimmunized with the purified fusion proteins reacted with serotype 1, MDV-infected CEF in the fluorescent antibody (FA) test to significant titers. These immune sera did not react with either serotype II or III, indicating the serotype specificity of the phosphorylated polypeptides.
 ...
PMID:Marek's disease virus gene clones encoding virus-specific phosphorylated polypeptides and serological characterization of fusion proteins. 169 56

We have studied the process of Moloney murine leukemia virus (M-MuLV) assembly by characterization of core (gag) protein mutants and analysis of wild-type (wt) gag proteins produced by cells in the presence of the ionophore monensin. Our genetic studies involved examination of linker insertion mutants of a Gag-beta-galactosidase (Gag-beta-gal) fusion protein, GBG2051, which is incorporated into virus particles when expressed in the presence of wt viral proteins. Analysis indicated that the amino-terminal two-thirds of the gag matrix domain is essential for targeting of proteins to the plasma membrane; mutant proteins localized to the cytoplasm or were trapped on intracellular membranes. Mutations through most of the coding region of the gag capsid domain generated proteins which were released from cells in membrane vesicles but not in virions. In contrast, linker insertions into p12gag or carboxy-terminal portions of the matrix or capsid coding regions did not affect assembly of fusion proteins into virus particles. Monensin, which blocks vesicular transport, inhibited gag protein intracellular transport and release from cells. Our results suggest that a significant proportion of M-MuLV myristylated gag proteins travel via vesicles to the cell surface. Specific matrix protein polypeptide regions and myristic acid modification are both necessary for appropriate gag protein transport, while capsid protein interactions appear to mediate the final phase of virion formation.
 ...
PMID:Transport and assembly of gag proteins into Moloney murine leukemia virus. 169 96

We have expressed a number of polypeptides derived from the capsid proteins of the human parvovirus B19 in Escherichia coli. These include native VP1 (84K) and VP2 (58K) proteins and also fusions to beta-galactosidase containing differing amounts of the amino terminus of the VP1/2 polypeptide. Although each of these was expressed at high levels and the majority were produced as full-length proteins, only one was soluble. This soluble polypeptide, p132, is a beta-galactosidase fusion protein that includes 145 amino acids from B19 which are entirely derived from the region unique to VP1. Despite containing such a small portion of VP1, which itself constitutes only 4% of total capsid protein, p132 reacted with all our known anti-B19 IgM-positive human serum samples. We conclude that this region contains epitopes which must be prominently exposed on the intact virus. We have demonstrated the use of this recombinant antigen in a simple diagnostic assay for B19-specific antibodies which can be used for initial screening of human serum samples. In a survey of 103 serum specimens, our ELISA positively identified all samples (19/19) which were positive by IgM antibody capture radioimmunoassay. The recombinant p132 antigen is efficiently produced and readily purified from E. coli, and its use as a diagnostic antigen should increase the availability of routine clinical testing for human parvovirus infection.
 ...
PMID:The production of human parvovirus capsid proteins in Escherichia coli and their potential as diagnostic antigens. 170 79

A 7.2 kb Bg/II restriction fragment, which increases the production of several extracellular enzymes, including alkaline phosphatase, amylase, protease, lipase and beta-galactosidase, was cloned in Streptomyces lividans from the DNA of S. griseus ATCC 10137. This gene (named saf) showed a positive gene dosage effect on production of extracellular enzymes. When the saf gene was introduced into cells in high copy numbers it delayed the formation of pigments and spores in S. lividans and also retarded actinorhodin production in Streptomyces coelicolor. The saf gene hybridized with specific bands in the DNA of several Streptomyces strains tested. A 1 kb fragment containing the saf gene was sequenced and contains an open reading frame (ORF) of 306 nucleotides which encodes a polypeptide of Mr 10,500. This ORF is contained within a fragment of 432 bp which retained activity in Streptomyces. A fragment with promoter activity is present upstream of the saf reading frame. The predicted Saf polypeptide has a strong positive charge, and does not show a typical amino acid composition for a membrane protein, and contains a DNA-binding domain similar to those found in several regulatory proteins.
 ...
PMID:Cloning and characterization of a gene of Streptomyces griseus that increases production of extracellular enzymes in several species of Streptomyces. 170 69


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>