Duellman Sarah J, Thompson Nancy E, Burgess Richard R
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI 53706, USA.
Protein Expr Purif. 2004 May;35(1):147-55. doi: 10.1016/j.pep.2003.12.011.
Polyol-responsive monoclonal antibodies (PR-mAbs) provide a strategy to purify active, nondenatured proteins by a single-step immunoaffinity chromatography procedure. The high affinity interaction between these antibodies and the antigen can be dissociated in the presence of a nonchaotropic salt and a low molecular weight polyhydroxylated compound (polyol). The epitope for PR-mAb IIB8 is located near the N-terminus of the human transcription factor IIB (TFIIB). The epitope is an eight amino acid sequence, TKDPSRVG, that can be fused to a desired protein for use as a purification tag. This epitope tag (termed hIIB) was fused to the C-terminus of green fluorescent protein (GFP). An additional GFP fusion protein utilized another version of hIIB containing a point mutation at position two. These fusion proteins, expressed in Escherichia coli, allowed successful separation of the desired protein in a single chromatographic step. This strategy extends PR-mAb gentle-release purification to numerous expressed proteins.
多元醇反应性单克隆抗体（PR-mAbs）提供了一种通过单步免疫亲和色谱法纯化活性、非变性蛋白质的策略。这些抗体与抗原之间的高亲和力相互作用可以在非离液盐和低分子量多羟基化化合物（多元醇）存在的情况下解离。PR-mAb IIB8的表位位于人转录因子IIB（TFIIB）的N端附近。该表位是一个八氨基酸序列TKDPSRVG，可与所需蛋白质融合用作纯化标签。这个表位标签（称为hIIB）与绿色荧光蛋白（GFP）的C端融合。另一种GFP融合蛋白使用了hIIB的另一个版本，该版本在第2位含有一个点突变。这些在大肠杆菌中表达的融合蛋白能够在单个色谱步骤中成功分离出所需蛋白质。这种策略将PR-mAb温和释放纯化扩展到众多表达的蛋白质。
