Goldsmith Moshe, Kiss Csaba, Bradbury Andrew R M, Tawfik Dan S
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
Protein Eng Des Sel. 2007 Jul;20(7):315-8. doi: 10.1093/protein/gzm026. Epub 2007 Jun 15.
This article describes a set of standard control experiments for the authentication of new protein variants isolated through library selection and site-directed mutagenesis. These controls are specifically designed to rule out artifacts derived from 'double transformants' -- i.e. cells transformed with, or infected by, two different plasmids simultaneously. These seem to have been the source of past artifacts and, as demonstrated here, are far more common than generally recognized. By following standard protocols for cloning, plasmid isolation, subcloning, in combination with functional assays, the presence of such artifacts can be ruled out. This protocol needs to be applied for any new variant isolated from heterogeneous gene repertoires, and in particular for variants isolated by selection for either enzymatic activity, or binding.
本文描述了一组标准对照实验，用于鉴定通过文库筛选和定点诱变分离得到的新蛋白质变体。这些对照实验专门设计用于排除源自“双转化体”的假象——即同时被两种不同质粒转化或感染的细胞。这些似乎是过去假象的来源，并且如本文所示，其出现频率比普遍认为的要高得多。通过遵循克隆、质粒分离、亚克隆的标准方案，并结合功能测定，可以排除此类假象的存在。对于从异质基因库中分离得到的任何新变体，特别是通过酶活性或结合筛选分离得到的变体，都需要应用此方案。
