Olchowy Jarosław, Kur Krzysztof, Sachadyn Paweł, Milewski Sławomir
Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of Technology, 11/12 Narutowicza St., 80-952 Gdańsk, Poland.
Protein Expr Purif. 2006 Apr;46(2):309-15. doi: 10.1016/j.pep.2005.07.030. Epub 2005 Aug 24.
Expression plasmids containing recombinant genes encoding three His(6)-tagged versions of the enzyme, glucosamine-6-phosphate synthase from Candida albicans, were constructed and overexpressed in Escherichia coli. The gene products were purified by metal-affinity chromatography to near homogeneity with 77-80% yield and characterized in terms of size and enzymatic properties. Presence of oligohistidyl tags at either of two ends did not affect enzyme quarternary structure but strongly influenced its catalytic activity. The His6-N-tagged enzyme completely lost an ability of glucosamine-6-phosphate formation and amidohydrolase activity but retained the hexosephosphate-isomerising activity. On the other hand, two His6-C-tagged versions of glucosamine-6-phosphate synthase exhibited amidohydrolase activity almost equal to that of the wild-type enzyme but only 18% of its hexosephosphate-isomerising activity and about 1.5% of the synthetic activity.
构建了含有编码白色念珠菌葡糖胺-6-磷酸合酶三种His(6)标签形式的重组基因的表达质粒，并在大肠杆菌中进行了过表达。通过金属亲和色谱法将基因产物纯化至接近均一，产率为77-80%，并对其大小和酶学性质进行了表征。在两端任何一端存在寡聚组氨酸标签均不影响酶的四级结构，但强烈影响其催化活性。His6-N标签化的酶完全丧失了形成葡糖胺-6-磷酸的能力和酰胺水解酶活性，但保留了己糖磷酸异构化活性。另一方面，葡糖胺-6-磷酸合酶的两种His6-C标签形式表现出几乎与野生型酶相等的酰胺水解酶活性，但仅具有其己糖磷酸异构化活性的18%和约1.5%的合成活性。
