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)

Escherichia coli LE392 (pAL28) was previously isolated as a positive clone harboring the alginate lyase gene (aly) from an alginate-degrading strain, Pseudomonas sp. OS-ALG-9. The plasmid pAL205, one of the constructs obtained after successive subcloning of pAL28, gave the highest expression of aly in E. coli cells. A 8-fold increase in the alginate lyase (Aly) activity in E. coli JM109 (pAL205) was induced with isopropyl-beta-D-thiogalactoside, which was 210 times higher than that in E. coli LE392 (pAL28). The highly significant increase in the expression of the Aly enzyme with pAL205 was investigated through the nucleotide sequence around the 5' region of aly as well as the N-terminal sequence of the purified enzyme. It was found that the Aly expressed in E. coli (pAL205) was a fused protein containing 7 residues from the N-terminus of beta-galactosidase alpha-peptide and the mature protein found in the Pseudomonas sp. except for three residues in the N-terminal.
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PMID:High gene expression in Escherichia coli of recombinant alginate lyase as a fused protein with beta-galactosidase alpha-peptide. 789 71

The plasmid pAL205 encodes an alginate lyase gene of Pseudomonas sp. OS-ALG-9, fused in frame to the beta-galactosidase alpha-peptide gene. The alginate lyase (Aly) expressed in Escherichia coli (pAL205) was significantly secreted into the medium by the addition of glycine. The extracellular enzyme isolated from the culture of E. coli JM109 (pAL205) was purified over 15,000-fold by successive chromatography and subjected to amino acid sequence analysis. The sequence determined was identical to that of the intracellular protein. Since the activity and molecular size of the extracellular Aly is identical to the intracellular protein and to the Aly isolated from Pseudomonas, the glycine does not affect or modify the Aly during its leakage into the medium.
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PMID:Purification and characterization of the recombinant alginate lyase from Pseudomonas sp. leaked by Escherichia coli upon addition of glycine. 789 72

Trials of gene transfer for cystic fibrosis (CF) are currently underway. However, direct application to the airways may be impeded by the presence of airway secretions. We have therefore assessed the effect of CF sputum on the expression of the reporter gene beta-galactosidase complexed with the cationic liposome DC-Chol/DOPE in a number of cell lines in vitro. Transfection was markedly inhibited in the presence of sputum; the effect was concentration dependent and was only partially ameliorated by removal of sputum with phosphate-buffered saline (PBS) washing before gene transfer. However, treatment of the sputum-covered cells with recombinant human DNase (rhDNase, 50 micrograms/ml) but not with N-acetylcysteine, Nacystelyn, lysine (all 20 mM) or recombinant alginase (0.5 U/ml) significantly (P < 0.005) improved gene transfer. Adenovirus-mediated gene transfer efficiency in the presence of sputum was similarly inhibited, and again, treatment with rhDNase before transfection significantly improved gene transfer (P < 0.005). Transfection of Cos 7 cells in the presence of exogenous genomic DNA alone demonstrated similar inhibition to that observed with sputum and was also ameliorated by pre-treatment of DNA-covered cells with rhDNase. In a separate series of experiments performed in the absence of added sputum or genomic DNA, increasing concentrations of rhDNase resulted in a concentration-related decline in transfection efficiency. However, even at the highest concentration (500 micrograms/ml of rhDNase), transfection efficiency remained more than 50% of control. Thus, pre-treatment of CF airways with rhDNase may be appropriate before liposome or adenovirus-mediated gene therapy.
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PMID:The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro. 953 69