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)

An infectious genome of the Junonia coenia densovirus (Jc DNV) has been recently cloned and sequenced. We investigated the ability of this cloned genome to be used as expression vector by inserting the lacZ gene of Escherichia coli as fusion gene in the major open reading frame (ORF 1) of the viral sequence. The resulting recombinant plasmid designated pBRJlac Z was transfected into insect SPC-SL 52 cells and the expression of beta-galactosidase (beta-gal) was detected qualitatively or quantitatively by using Xgal or ONPG as chromogenic substrates. Western blot analysis revealed that beta-gal was expressed as chimeric capsid-beta-gal polypeptides. This provided evidence that ORF1 codes for structural polypeptides which share a common C-terminal sequence. Construction of plasmids with alterations or deletions in ORF2, 3 or 4, allowed us to implicate nonstructural (NS) functions in viral DNA replication. Deletions in inverted terminal repeats or in NS functions did not abolish expression of capsid polypeptides but reduced it dramatically. Encapsidation of Jlac Z recombinant genome was achieved by trans-complementation with plasmids bearing intact structural and nonstructural functions. Detection of a beta-gal activity in SPC-SL 52 cells following several subcultures post-transfection suggests that Jlac Z recombinant genome could be maintained in an integrative or episomal state.
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PMID:The densovirus of Junonia coenia (Jc DNV) as an insect cell expression vector. 153 Jul 77

Strategies for the expression of precursors of eukaryotic secreted proteins as part of fused proteins in Escherichia coli have been explored. A fusion protein with beta-galactosidase at the N-terminal end and honeybee prepromelittin at the C-terminal end (beta-gal-pM) was expressed in low amounts as a cleaved polypeptide, from which the promelittin portion had been removed. Inclusion in the induction culture of 10 mM MgCl2 or 8.3% (v/v) ethanol, inhibitors of signal peptidase, gave rise to the full-length beta-gal-pM fusion protein. The results suggest that a soluble recombinant fusion protein with a signal peptide in an internal location 660 residues from the N-terminus is recognized by the E. coli translocation apparatus in the inner membrane and by leader peptidase. High-level production (about 45% of total cellular proteins) of prepromelittin was achieved when it was part of a fusion protein at the C-terminus of a truncated insoluble polypeptide from bacteriophage gene 10. This fusion protein separated into inclusion bodies in an aggregated form. In contrast, attempts to express prepromelittin by itself or at the N-terminal end of a fusion with mouse dihydrofolate reductase (pM-DHFR) proved unsuccessful.
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PMID:Expression of honeybee prepromelittin as a fusion protein in Escherichia coli. 182 10

cDNA encoding human preproPTH (hpreproPTH) was expressed in Escherichia coli to study the processing of the precursor to hPTH and its secretion by the bacterial secretory apparatus. We first constructed hybrid genes that differed randomly in the distance between the E. coli lac promoter's ribosomal binding site and DNA encoding a fusion protein with beta-galactosidase activity and the prepro sequence of hpreproPTH on the aminoterminus. Starting with clones identified as efficient producers of beta-galactosidase on indicator agar plates, the coding sequence for hpreproPTH was reconstituted intact. In a different construction we placed the hpreproPTH coding sequence downstream from the lac promoter at a distance of 12 base pairs from the ribosomal binding site. PTH immunoreactive proteins from multiple clones were identified by protein gel electrophoresis and by protein microsequencing. PTH-related proteins encoded by different plasmids were shown to be hpreproPTH with amino-terminal extensions of either two or four amino acids and as authentic hpreproPTH. Two hPTH fragments, hPTH(3-84) and hPTH(8-84), were also observed. The trypsin accessibility of hpreproPTH and of the two hPTH fragments in pulse-chase, cell-fractionation experiments using intact and lysed spheroplasts lets us conclude that the mammalian signal sequence directs hpreproPTH to the surface of the spheroplast membrane but is not appropriately cleaved by the signal peptidase.
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PMID:Human preproparathyroid hormone synthesized in Escherichia coli is transported to the surface of the bacterial inner membrane but not processed to the mature hormone. 333 11

An alpha-neo-endorphin (alpha NE) gene, which we previously synthesized chemically and inserted into E. coli beta-galactosidase gene of pK013 plasmid, has been excised and fused to E. coli alkaline phosphatase (APase) gene. One of the transformants was named E15/pA alpha NE1. Under the APase gene regulation, APase-alpha NE chimeric protein was expressed at 1.3 X 10(6) molecules per cell, and accounted for about 60% of total cellular proteins. The HPLC pattern of CNBr treated E15/pA alpha NE1 was very simple reflecting the high content of the chimeric protein and low numbers of methionine residues in it. A series of genes encoding APase-alpha NE chimeric proteins in which 30 to 94 C-terminal amino acid residues were replaced by (met)-alpha NE, was cloned in E. coli. Transportation of the chimeric proteins to periplasmic space was studied. All chimeric proteins were apparently processed by signal peptidase but few, if any, was transported to the periplasmic space.
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PMID:Expression of chemically synthesized alpha-neo-endorphin gene fused to E. coli alkaline phosphatase. 629 40

The core protein of hepatitis C virus (HCV) is considered to be cleaved from the N terminus of the large precursor polyprotein by cellular signalase. The HCV cDNA encoding the core protein was expressed (i) in monkey COS cells by a plasmid expression vector driven by the SR alpha promoter, and (ii) in insect cells by a recombinant baculovirus. The expressed product had an M(r) of 22,000 and was located in the cytoplasm. When the C-terminal hydrophobic domains were deleted, however, the truncated core proteins were translocated into the nucleus. The truncated core proteins were located in the nucleus even when they were expressed as a fusion protein with E. coli beta-galactosidase, which is essentially localized in the cytoplasm. Plasmids containing HCV cDNAs with a deletion in one of the regions encoding clusters of basic amino acids were expressed in COS cells and the localization of the core protein was examined. The residues PRRGPR were suggested to play an important role in nuclear localization. HCV is an RNA virus and its life cycle was originally considered to be confined to the cytoplasm; the present study, however, suggests that the HCV core protein can translocate into the nucleus under certain circumstances.
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PMID:Nuclear localization of the truncated hepatitis C virus core protein with its hydrophobic C terminus deleted. 784 42

The aim of this work was to express a eukaryotic pre-protein in Escherichia coli so that it could be obtained intact, without cleavage, by bacterial leader peptidase. To this end, cDNA coding for honeybee prepromelittin was ligated to the 3' end of genes coding for truncated forms of either Protein A or beta-galactosidase (beta-Gal) under the control of inducible promoters, with an oligonucleotide coding for the Factor Xa cleavage site at the junction between the two proteins. The Protein A fusion was expressed in good yield, and about 80% of it formed inclusion bodies. The prepromelittin section of the Protein A fusion caused some export of the intact fusion protein to the growth medium. The prepromelittin beta-Gal fusion was expressed in low yield and became associated with the E. coli cytoplasmic membrane. Its expression was toxic to E. coli. Thus, the synthesis of a full-length eukaryotic pre-protein in E. coli is best achieved when the fusion protein forms inclusion bodies.
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PMID:Expression and export in Escherichia coli of fusion proteins containing carboxy-terminally located honeybee prepromelittin. 806 10

The removal of the signal peptide from a precursor protein is a crucial step of protein secretion. In order to improve Bacillus megaterium as protein production and secretion host, the influence of homologous type I signal peptidase SipM overproduction on recombinant Leuconostoc mesenteroides dextransucrase DsrS synthesis and export was investigated. The dsrS gene was integrated as a single copy into the chromosomal bgaM locus encoding beta-galactosidase. Desired clones were identified by blue-white selection. In this strain, the expression of sipM from a multicopy plasmid using its own promoter increased the amount of secreted DsrS 3.7-fold. This increase in protein secretion by SipM overproduction was next transferred to a high level DsrS production strain using a multicopy plasmid encoding sipM with its natural promoter and dsrS under control of a strong xylose-inducible promoter. No further increase in DsrS export were observed when this vector was carrying two sipM copies. Similarly, bicistronic sipM and dsrS high level expression did not enhance DsrS secretion, indicating the natural limitation of the approach. Interestingly, SipM-enhanced DsrS secretion also resulted in an overall increase of DsrS production.
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PMID:Coexpression of the type I signal peptidase gene sipM increases recombinant protein production and export in Bacillus megaterium MS941. 1600 78

The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The beta-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.
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PMID:Signal recognition particle-dependent inner membrane targeting of the PulG Pseudopilin component of a type II secretion system. 1715 57

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma, a transmissible lung cancer in sheep. Previous experiments in differentiated murine tissue culture cell lines suggested that the disease specificity of JSRV for secretory lung epithelial cells (type II pneumocytes an Clara cells) reflects transcriptional specificity of the viral long terminal repeat (LTR) for these cells. To test this in vivo, transgenic mice carrying the bacterial beta-galactosidase (beta-Gal) gene driven by the JSRV LTR were generated. Two transgenic lines showed beta-Gal expression in the lungs but not other tissues of F1 animals, although transgene silencing in subsequent generations was a major problem. The cells expressing the transgene were identified by two- and three-color immunofluorescence for marker proteins of type II pneumocytes (surfactant protein C [SPC]) and Clara cells (CC10) as well as for a T7 gene 10 epitope present in the beta-Gal reporter. F1 animals from both lines showed transgene expression in type II pneumocytes, but somewhat surprisingly not in Clara cells. Expression was not detected in bronchiolo-alveolar stem cells (BASCs) either. These results indicate that the JSRV LTR is specifically active in type II pneumocytes in the mouse lung, which is consistent with the fact that JSRV-induced OPA tumors in sheep largely have phenotypic markers of type II pneumocytes.
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PMID:Specific in vivo expression in type II pneumocytes of the Jaagsiekte sheep retrovirus long terminal repeat in transgenic mice. 1805 63