Denman J, Hayes M, O'Day C, Edmunds T, Bartlett C, Hirani S, Ebert K M, Gordon K, McPherson J M
Genzyme Corporation, Framingham, MA 01701.
Biotechnology (N Y). 1991 Sep;9(9):839-43. doi: 10.1038/nbt0991-839.
A glycosylation variant of human tissue-type plasminogen activator (tPA) designated longer-acting tissue-type plasminogen activator (LAtPA) was extensively purified from the milk of a transgenic goat by a combination of acid fractionation, hydrophobic interaction chromatography and immunoaffinity chromatography. This scheme provided greater than 8,000-fold purification of the protein, a cumulative yield of 25% and purity greater than 98% as judged by SDS gel electrophoresis. SDS gel electrophoresis revealed that the transgenic enzyme was predominantly the "two chain" form of the protease. The specific activity of the purified transgenic protein, based on the average of the values obtained for three different preparations, was 610,000 U/mg as judged by amidolytic activity assay. This was approximately 84% of the value observed for the recombinant enzyme produced in mouse C127 cells. Analysis of the transgenic protein indicated that it had a significantly different carbohydrate composition from the recombinant enzyme produced in C127 cells. Molecular size analysis of the oligosaccharides from the transgenic and C127 cell-derived LAtPA preparations confirmed their differences and showed that the mouse cell-derived preparation contained larger, complex-type N-linked oligosaccharide structures than the material produced in goat mammary tissue.
一种名为长效组织型纤溶酶原激活剂（LAtPA）的人组织型纤溶酶原激活剂（tPA）糖基化变体，通过酸分级分离、疏水相互作用色谱和免疫亲和色谱相结合的方法，从转基因山羊的乳汁中进行了广泛纯化。该方案实现了该蛋白超过8000倍的纯化，累积产率为25%，经SDS凝胶电泳判断纯度大于98%。SDS凝胶电泳显示，转基因酶主要为蛋白酶的“双链”形式。根据三种不同制剂获得的值的平均值判断，纯化的转基因蛋白的比活性通过酰胺分解活性测定为610,000 U/mg。这约为在小鼠C127细胞中产生的重组酶所观察到的值的84%。对转基因蛋白的分析表明，其碳水化合物组成与在C127细胞中产生的重组酶有显著差异。对来自转基因和C127细胞来源的LAtPA制剂的寡糖进行分子大小分析，证实了它们的差异，并表明小鼠细胞来源的制剂比山羊乳腺组织中产生的物质含有更大的、复合型N-连接寡糖结构。
