Bichet P, Mollat P, Capdevila C, Sarubbi E
Biotechnology Department, Hoechst Marion Roussel, Romainville, 93235, France.
Protein Expr Purif. 2000 Jun;19(1):197-201. doi: 10.1006/prep.2000.1239.
After affinity purification on immobilized glutathione, insect-cell-derived glutathione S-transferase (GST) fusion proteins contain variable amounts of protein contaminants of about 23-24 kDa. We have isolated these glutathione-binding proteins from the widely used Sf9 and Hi5 insect cell lines and characterized them by LC-MS and N-terminal sequencing. Based on the observation that these proteins have higher affinity for glutathione than GST fusions, we have found that by using differential elution conditions the amount of such contaminants in GST fusion preparations can be strongly reduced directly during the affinity purification step. The main interest of these results is that they are not restricted to a specific construct, but rather they seem to apply to various insect-cell-derived GST fusions.
在固定化谷胱甘肽上进行亲和纯化后，昆虫细胞衍生的谷胱甘肽S-转移酶（GST）融合蛋白含有可变数量的约23 - 24 kDa的蛋白质污染物。我们从广泛使用的Sf9和Hi5昆虫细胞系中分离出这些谷胱甘肽结合蛋白，并通过液相色谱-质谱联用（LC-MS）和N端测序对其进行了表征。基于这些蛋白质对谷胱甘肽的亲和力高于GST融合蛋白这一观察结果，我们发现通过使用差异洗脱条件，在亲和纯化步骤中可以直接大幅减少GST融合制剂中此类污染物的含量。这些结果的主要意义在于，它们并不局限于特定的构建体，而是似乎适用于各种昆虫细胞衍生的GST融合蛋白。
