Department of Biotechnology and Molecular Sciences, University of Insubria, Varese, Italy.
Biotechnol Appl Biochem. 2011 Jan-Feb;58(1):39-49. doi: 10.1002/bab.7.
We report the expression of recombinant RNASET2, the only human member of the Rh/T2/S family of acid ribonucleases, in the yeast Pichia pastoris and the baculovirus-insect cell heterologous systems. In both models, the yield of recombinant protein was comparable and ranged between 5 mg/L (for a catalytically impaired mutant version of RNASET2) and 30 mg/L for the wild-type protein. Thus, the produced protein version rather than the expression system used appears to influence protein yield after optimization of culture conditions. The recombinant protein was found to undergo heterogeneous glycosylation in both systems, particularly in P. pastoris. Most importantly, the wild-type protein purified from both systems was found to be catalytically competent. The expression of recombinant RNASET2 in both systems will allow the implementation of functional assays in vivo and in vitro to better define the antioncogenic properties of this member of the Rh/T2/S ribonuclease family.
我们报告了重组 RNASET2 的表达，这是 Rh/T2/S 酸性核糖核酸酶家族中唯一的人类成员，在酵母巴斯德毕赤酵母和杆状病毒-昆虫细胞异源系统中。在这两种模型中，重组蛋白的产量相当，范围在 5mg/L（对于催化功能丧失的 RNASET2 突变体版本）和 30mg/L 之间，对于野生型蛋白。因此，在优化培养条件后，似乎是所产生的蛋白版本而不是使用的表达系统影响蛋白产量。在这两种系统中，重组蛋白都发现经历了不均一的糖基化，尤其是在巴斯德毕赤酵母中。最重要的是，从这两种系统中纯化的野生型蛋白被发现具有催化活性。在这两种系统中表达重组 RNASET2 将允许在体内和体外实施功能测定，以更好地定义 Rh/T2/S 核糖核酸酶家族这一成员的抗癌特性。
