Thompson N E, Burgess R R
McArdle Laboratory for Cancer Research, University of Wisconsin at Madison 53706.
Protein Expr Purif. 1994 Oct;5(5):468-75. doi: 10.1006/prep.1994.1066.
The human RNA polymerase II transcription factor IIB (TFIIB) was purified from a bacterial expression system by immunoaffinity chromatography. Mouse monoclonal antibodies (MAbs) were prepared that react with TFIIB. A modified enzyme-linked immunosorbent assay was used to screen for MAbs that release the antigen in the presence of a low molecular weight polyhydroxylated compound and a nonchaotropic salt (polyol-responsive MAbs). One polyol-responsive MAb (designated IIB8) was purified by chromatography on protein A and conjugated to cyanogen bromide-activated Sepharose 4B. Escherichia coli strain BL21 (DE3) containing the pLysS plasmid was transformed with the human TFIIB gene contained in the pET11a vector (phIIB). After induction with IPTG, the cells were harvested and lysed. The lysate was treated with 0.5% polyethyleneimine and centrifuged. The supernatant fluid was applied to the IIB8-Sepharose. After extensive washing, the TFIIB was eluted with Tris-EDTA buffer containing 0.75 M ammonium sulfate and 40% propylene glycol. The purified TFIIB was active when added back to TFIIB-depleted HeLa nuclear extract and when used in the IgH minimal promoter system. This method will be useful for the rapid purification of TFIIB mutants and for the purification of large amounts of highly purified TFIIB for biochemical studies. In addition, this procedure establishes the general applicability of the use of polyol-responsive MAbs for the rapid, gentle purification of labile proteins.
人RNA聚合酶II转录因子IIB（TFIIB）通过免疫亲和层析从细菌表达系统中纯化得到。制备了与TFIIB反应的小鼠单克隆抗体（MAb）。使用改良的酶联免疫吸附测定法筛选在低分子量多羟基化化合物和非离液盐存在下能释放抗原的MAb（多元醇反应性MAb）。一种多元醇反应性MAb（命名为IIB8）通过蛋白A层析纯化，并与溴化氰活化的琼脂糖4B偶联。用包含在pET11a载体（phIIB）中的人TFIIB基因转化含有pLysS质粒的大肠杆菌菌株BL21（DE3）。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导后，收获细胞并裂解。裂解物用0.5%聚乙烯亚胺处理并离心。将上清液应用于IIB8-琼脂糖。经过大量洗涤后，用含有0.75 M硫酸铵和40%丙二醇的Tris-EDTA缓冲液洗脱TFIIB。纯化的TFIIB重新添加到TFIIB缺失的HeLa细胞核提取物中以及用于IgH最小启动子系统时具有活性。该方法将有助于快速纯化TFIIB突变体以及纯化大量用于生化研究的高纯度TFIIB。此外，该程序确立了使用多元醇反应性MAb快速、温和地纯化不稳定蛋白质的普遍适用性。
