PubMed:10024475 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PubMed/sourceid/10024475","sourcedb":"PubMed","sourceid":"10024475","source_url":"http://www.ncbi.nlm.nih.gov/pubmed/10024475","text":"Optimized heterologous expression of glutathione reductase from Cyanobacterium anabaena PCC 7120 and characterization of the recombinant protein.\nGlutathione reductase (GR) from the cyanobacterium Anabaena PCC 7120 was heterologously expressed in Escherichia coli SG5. Silent random mutations were introduced in the 5' region of DNA encoding the enzyme in order to generate a high-level expression clone. To maximize protein expression, the culture conditions were also optimized. In the high-level expression clones selected, E. coli-preferred codons were selectively used at certain positions. Under the optimal expression conditions, a yield of 17 mg recombinant protein per liter was obtained, which is about 10-fold higher than that of the wild-type enzyme. A hexahistidine tag was added at the C-terminal of the protein in order to allow IMAC affinity purification. This strategy simplified the purification process and provided a homogeneous enzyme for functional characterization. Anabaena GR uses NADPH as a coenzyme, like most of the GRs from other sources, but the KM values for NADPH and GSSG are higher than those of enzymes previously studied. The Anabaena enzyme also shows significant activity when NADH is used as a reductant.","tracks":[{"project":"CyanoBase","denotations":[{"id":"T1","span":{"begin":37,"end":58},"obj":"protein"},{"id":"T2","span":{"begin":146,"end":167},"obj":"protein"},{"id":"T3","span":{"begin":169,"end":171},"obj":"protein"},{"id":"T4","span":{"begin":998,"end":1000},"obj":"protein"}],"attributes":[{"subj":"T1","pred":"source","obj":"CyanoBase"},{"subj":"T2","pred":"source","obj":"CyanoBase"},{"subj":"T3","pred":"source","obj":"CyanoBase"},{"subj":"T4","pred":"source","obj":"CyanoBase"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"CyanoBase","color":"#93b6ec","default":true}]}]}}