PMID:3047129
| Citation |
Yu, F, Jen, Y, Takeuchi, E, Inouye, M, Nakayama, H, Tagaya, M and Fukui, T (1988) Alpha-glucan phosphorylase from Escherichia coli. Cloning of the gene, and purification and characterization of the protein. J. Biol. Chem. 263:13706-11 |
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| Abstract |
By using a synthetic oligonucleotide probe identical to a part of the gene for the Escherichia coli major outer membrane lipoprotein, we have cloned a gene from E. coli chromosomal DNA. However, the cloned gene was not one of the lipoprotein genes. The amino acid sequence deduced from its nucleotide sequence shows extensive similarities instead to alpha-glucan phosphorylase (EC 2.4.1.1). The gene, glgP, is located immediately downstream from glgA, the gene for glycogen synthase. The glgP gene was inserted into pUC9 vector and expressed in the presence of the lac inducer. The gene product was purified to apparent homogeneity as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In all chromatographies, the protein was eluted accompanied by a low phosphorylase activity. The final preparation showed phosphorolytic activity to various alpha-glucans, although the specific activity was extremely low compared to other alpha-glucan phosphorylases under the standard assay conditions. Its enzymatic activity, however, increased almost linearly as the concentration of glucan increased, reaching a value comparable with those of other phosphorylases. The amino acid sequence deduced was compared with those of alpha-glucan phosphorylases from other sources. |
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| Keywords |
Amino Acid Sequence; Base Sequence; Chromatography; Cloning, Molecular; DNA, Bacterial/genetics; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Escherichia coli/genetics; Genes, Bacterial; Molecular Sequence Data; Molecular Weight; Phosphorylases/genetics; Phosphorylases/isolation & purification; Phosphorylases/metabolism; Plasmids; Substrate Specificity |
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