Anderson M, Blowers D, Hewitt N, Hedge P, Breeze A, Hampton I, Taylor I
Cancer & Infection Research Departments, Protein Structure Laboratory, Zeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, United Kingdom.
Protein Expr Purif. 1999 Mar;15(2):162-70. doi: 10.1006/prep.1998.0996.
This report describes the cloning of recombinant human Bcl-2, in which the putative disordered loop region has been replaced with a flexible linker and the hydrophobic C-terminus has been replaced with a 6xHis tag (Bcl-2(6-32)-AAAA-Bcl-2(86-206)-HHHHHH, abbreviation rhBcl-2; amino acid numbering excludes the initiating methionine). This protein was expressed in Escherichia coli where it accumulated in insoluble form in inclusion bodies. After lysis the washed inclusion bodies were solubilized and an l-arginine assisted protein refolding route was employed to obtain biologically active protein. rhBcl-2 was purified further by nickel chelate chromatography to give protein of >95% purity, with an overall yield of 5 mg per g of E. coli cell paste. Edman sequencing showed that approximately 90% of the rhBcl-2 retained the initiating methionine residue. Analytical size exclusion chromatography suggested that the refolded and purified rhBcl-2 was monomeric in nondenaturing solution. Purified protein had an affinity for a Bax BH3 domain peptide comparable to that for in vivo folded recombinant human Bcl-2 and suppressed caspase activation in a cell-free assay for apoptosis. 1H NMR spectroscopy of rhBcl-2, both free and complexed with the Bax BH3 domain peptide, provided further evidence for the structural and functional integrity of the refolded protein. These findings parallel and extend those of Muchmore et al., who found that a loop deletion mutant of human Bcl-XL retained anti-apoptotic function.
本报告描述了重组人Bcl-2的克隆，其中假定的无序环区被柔性接头取代，疏水C末端被6xHis标签取代（Bcl-2(6 - 32)-AAAA-Bcl-2(86 - 206)-HHHHHH，缩写为rhBcl-2；氨基酸编号不包括起始甲硫氨酸）。该蛋白在大肠杆菌中表达，以不溶性形式聚集在包涵体中。裂解后，洗涤过的包涵体被溶解，并采用L-精氨酸辅助的蛋白质重折叠途径获得具有生物活性的蛋白。rhBcl-2通过镍螯合层析进一步纯化，得到纯度>95%的蛋白，每克大肠杆菌细胞糊的总产率为5毫克。埃德曼测序表明，约90%的rhBcl-2保留了起始甲硫氨酸残基。分析型尺寸排阻色谱表明，重折叠和纯化后的rhBcl-2在非变性溶液中为单体。纯化后的蛋白对Bax BH3结构域肽的亲和力与体内折叠的重组人Bcl-2相当，并在无细胞凋亡检测中抑制了半胱天冬酶的激活。rhBcl-2（游离状态以及与Bax BH3结构域肽复合状态）的1H NMR光谱为重折叠蛋白的结构和功能完整性提供了进一步证据。这些发现与Muchmore等人的研究结果相似且有所扩展，他们发现人Bcl-XL的环缺失突变体保留了抗凋亡功能。
