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)

A radio-labeled plasmid pTracer/Bsd/LacZ containing LacZ reporter gene was complexed with different molecular weights of chitosans (CS). Mouse myoblast cell line C2C12 was transfected by these chitosan-plasmid DNA complexes, and lipofectamine 2000 was used as control. Forty-eight hours after transfection, the activity of beta-galactosidase and radioactive count of cell lysis were determined. It was found that chitosan, especially low molecular weight species, had a surprising ability to deliver DNA into cells, since the radioactive count of cells transfected by chitosan-DNA complexes was even two times that of cells transfected by lipofectamine 2000. But the beta-galactosidase activity of chitosan/DNA complexes was much lower compared to that of lipofectamine 2000. Chitosanase which could degrade chitosan in specific mode was transported into C2C12 cells by osmotic lysis prior to gene delivery. Then these chitosanase-modified cells were transfected by CS-DNA complexes. The results indicated that beta-galactosidase activity in these cells increased markedly to 425.4 +/- 45.1 U/mg protein, nearly two-fold as that of cells transfected by liposome. This transfection protocol was also applied to 3T3 mouse fibroblast, 2T3 mouse osteoblast and MG63 human osteosarcoma cell lines, and an increased gene expression level was observed without exception. It is thought that the incorporated chitosanase could aid in chitosan degradation, which would promote gene unpacking, consequently increasing gene expression.
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PMID:Pre-deliver chitosanase to cells: a novel strategy to improve gene expression by endocellular degradation-induced vector unpacking. 1656 55

Previously, we had demonstrated that insufficient intracellular unpacking of exogene from its chitosan carrier contributes towards the restricted transfection efficiency of CS/DNA complex. In order to enhance intracellular unpacking and thus improve the transfection efficiency, our present work has addressed a novel strategy of chitosanase gene (csn) co-transfection. An Aspergillus fumigatuscsn gene was semi-synthesized and cloned into a prokaryotic expression vector, plasmid pGEX-3X, meanwhile a mutant csn gene encoding an inactive Asp129-Asn chitosanase was generated by site-directed mutagenesis. Both active csn (acCSN) and inactive csn (inCSN) genes were expressed in bacteria cells and chitosan degradation activities of those purified recombinant proteins were tested. These csn genes were further subcloned into an eukaryotic expression vector, plasmid pTracer-CMV/Bsd, containing a gfp reporter gene. Recombinant plasmid pTracer-accsn or pTracer-incsn was co-transfected with plasmid pTracer/Bsd/LacZ, which contains an additional lacZ reporter gene, into C2C12 myoblast cells by CS/DNA complex. The expression of gfp reporter gene was determined by fluorescence microscope, while the expression of lacZ reporter was evaluated quantitatively by beta-galactosidase activity. All together, findings indicate that during the exogene being delivered into mammalian cells by CS/DNA complex, the csn co-transfection is beneficial for the exogene expression.
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PMID:Improved transfection efficiency of CS/DNA complex by co-transfected chitosanase gene. 1806 72

Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with beta-galactosidase activity (Escherichia coli LacZ), beta-glucuronidase activity (Homo sapiens GusB), and beta-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-beta-D-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 A resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role of E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural beta-1,4-D-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-beta-D-glucosaminide synthetic substrate provide insight into interactions in the +1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.
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PMID:The structural basis of substrate recognition in an exo-beta-D-glucosaminidase involved in chitosan hydrolysis. 1897 64