Firek S, Whitelam G C, Draper J
Department of Botany, University of Leicester, UK.
Transgenic Res. 1994 Sep;3(5):326-31. doi: 10.1007/BF01973593.
We have investigated targeting to the endoplasmic reticulum (ER) of wild-type GUS and a modified form (GUS S358) by making an N-terminal fusion of the beta-glucuronidase (GUS) enzyme with the wheat alpha-amylase signal peptide. In vitro studies demonstrated that the modified GUS (S358) lacked the glycosylation site present within the wild-type enzyme. Analysis of transgenic tobacco plants revealed that the modified GUS enzyme retained activity upon passage to the ER. When further experiments were carried out to determine the cellular location of the modified GUS enzyme, it was found that (contrary to expectation) the majority of GUS activity was retained within the cell and was not secreted to the cell surface via the default pathway. The data indicated that the modified GUS enzyme is an unsuitable reporter enzyme for studying protein secretion.
我们通过将β-葡萄糖醛酸酶（GUS）与小麦α-淀粉酶信号肽进行N端融合，研究了野生型GUS和一种修饰形式（GUS S358）在内质网（ER）的靶向作用。体外研究表明，修饰后的GUS（S358）缺乏野生型酶中存在的糖基化位点。对转基因烟草植株的分析表明，修饰后的GUS酶在内质网传递后仍保留活性。当进一步进行实验以确定修饰后的GUS酶的细胞定位时，发现（与预期相反）大部分GUS活性保留在细胞内，并未通过默认途径分泌到细胞表面。数据表明，修饰后的GUS酶不适用于研究蛋白质分泌的报告酶。
