Centre for Synthesis and Chemical Biology, UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland.
Mol Biotechnol. 2012 Nov;52(3):244-50. doi: 10.1007/s12033-012-9542-7.
The EE subunit of horse liver alcohol dehydrogenase (HLADH-EE) has been subcloned in pRSETb vector to generate a fusion His-tag protein. The migration from a multistep purification protocol for this well-known enzyme to a single-step has been successfully achieved. Several adjustments to the traditional purification procedure for His-tag proteins have been made to retain protein activity. A full characterization of the fusion enzyme has been carried out and compared with the native one. The K (m) for EtOH, NAD and NADH in the His-tag version of HLADH are in line with the ones reported in literature for the native enzyme. A shift in optimal pH activity is also observed. The enzyme retains the same stability and quaternary structure as the wild type and can therefore be easily used instead of the native HLADH for biotechnological applications.
马肝醇脱氢酶（HLADH-EE）的 EE 亚基已被克隆到 pRSETb 载体中，以生成融合 His 标签蛋白。已成功地将该知名酶的多步纯化方案转化为单步纯化方案。对传统的 His 标签蛋白纯化程序进行了多项调整，以保留蛋白质活性。已对融合酶进行了全面表征，并与天然酶进行了比较。His 标签 HLADH 的 EtOH、NAD 和 NADH 的 K（m）与文献中报道的天然酶一致。还观察到最佳 pH 活性的偏移。该酶保留与野生型相同的稳定性和四级结构，因此可轻松替代天然 HLADH 用于生物技术应用。
