Barinova K V, Eldarov M A, Khomyakova E V, Muronetz V I, Schmalhausen E V
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119234, Russia; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119234, Russia.
Bioengineering Center of Russian Academy of Sciences, Prosp. 60-Letiya Oktyabrya 7/1, Moscow, 117312, Russia.
Protein Expr Purif. 2017 Sep;137:1-6. doi: 10.1016/j.pep.2017.06.009. Epub 2017 Jun 15.
The goal of the present work was expression of human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) without additional tag constructions in E. coli cells and elaboration of the procedure for purification of untagged hGAPDH from the extract of the producer cells. We present a simple method for purification of untagged hGAPDH including ammonium sulfate fractionation and gel filtration on a G-100 Sephadex column. The method allows isolation of 2 mg of pure hGAPDH from 600 ml of cell culture (7 g of the cell biomass). The specific activity of the freshly purified hGAPDH constitutes 117 ± 5 μmol NADH/min per mg protein (pH 9.0, 22 °C), which is close to the specific activity of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase determined under the same conditions and several times exceeds the specific activity of his-tagged GAPDH preparations. The high enzymatic activity suggests that the recombinant enzyme retains its native structure. The described procedure may be useful for researchers who need a preparation of native hGAPDH without admixture of misfolded forms for their investigations.
本研究的目标是在大肠杆菌细胞中表达无额外标签构建的人甘油醛-3-磷酸脱氢酶（hGAPDH），并制定从生产细胞提取物中纯化无标签hGAPDH的方法。我们提出了一种纯化无标签hGAPDH的简单方法，包括硫酸铵分级分离和在G-100葡聚糖凝胶柱上进行凝胶过滤。该方法可从600毫升细胞培养物（7克细胞生物量）中分离出2毫克纯hGAPDH。新鲜纯化的hGAPDH的比活性为每毫克蛋白质117±5微摩尔NADH/分钟（pH 9.0，22°C），这与在相同条件下测定的兔肌肉甘油醛-3-磷酸脱氢酶的比活性相近，并且比带His标签的GAPDH制剂的比活性高出几倍。高酶活性表明重组酶保留了其天然结构。所描述的方法可能对那些需要制备无错误折叠形式混合物的天然hGAPDH用于研究的研究人员有用。
