Kroiher M, Raffioni S, Steele R E
Department of Biological Chemistry, University of California, Irvine 92717-1700, USA.
Biochim Biophys Acta. 1995 Jul 3;1250(1):29-34. doi: 10.1016/0167-4838(95)00060-8.
The construction and use of a plasmid which allows the expression and single step purification of recombinant rat basic fibroblast growth factor (bFGF) is described. A cDNA encoding rat bFGF was subcloned into the expression plasmid pQE-9 (Qiagen) in such a way that the bFGF which is produced from the resulting construct contains 6 histidine residues near the amino terminus. The resulting plasmid, pQE-9-bFGF, was expressed in the E. coli strain M15[pREP4] and the 6 x His-bFGF was purified to homogeneity from the soluble fraction of the bacterial cell lysate in a single step by affinity chromatography on a nickel chelate resin. About 5 mg of 6 x His-bFGF was obtained from the soluble fraction from one liter of bacterial cell culture. Testing of the 6 x His-bFGF in a PC12 cell differentiation assay showed that its activity was comparable to the activities for native bFGF and recombinant bFGF purified by multistep methods.
本文描述了一种质粒的构建和使用方法，该质粒可用于重组大鼠碱性成纤维细胞生长因子（bFGF）的表达和一步纯化。将编码大鼠bFGF的cDNA亚克隆到表达质粒pQE-9（Qiagen公司）中，使得从所得构建体产生的bFGF在氨基末端附近含有6个组氨酸残基。所得质粒pQE-9-bFGF在大肠杆菌菌株M15[pREP4]中表达，通过镍螯合树脂亲和层析从细菌细胞裂解物的可溶部分中一步纯化出6×His-bFGF至均一性。从一升细菌细胞培养物的可溶部分中获得了约5mg的6×His-bFGF。在PC12细胞分化试验中对6×His-bFGF进行测试，结果表明其活性与天然bFGF和通过多步方法纯化的重组bFGF的活性相当。
