利用凝胶过滤层析技术分离人源γ-微管环形复合物

摘要

目的：分离人源γ-微管蛋白环形复合物（γ-tubulin ring complex ，γTuRC）。方法利用凝胶过滤层析技术分离人源293 FT细胞裂解产物；利用免疫印迹检测γTuRC和γ-微管蛋白小复合物（γ-tubulin small complex ，γTuSC）所在的洗脱组分，实现复合物与游离蛋白分离。结果γTuRC复合物组成型组分γ-微管蛋白（γ-tubulin）、γ-微管蛋白复合物蛋白2（γ-tubulin complex protein 2， GCP2）、GCP3和GCP4在第4洗脱组分（4＃）被洗脱富集，该组分相对分子质量约为2×106；γTuSC复合物组成型组分γ-tubulin、GCP2、GCP3蛋白在14＃洗脱组分被洗脱富集，该组分相对分子质量约为3．1×105；未掺入的游离蛋白组分在18＃洗脱组分后被洗脱下来。非变性凝胶电泳可在相应组分中检测到γTuRC和γTuSC复合物。结论γTuRC复合物在凝胶过滤层析4＃洗脱组分得到富集；γTuSC复合物在14＃洗脱组分得到富集。上述复合物与游离蛋白成功分离，可用于后续复合物组装研究。%Objective To separate human γ-tubulin ring complexes (γTuRC) .Methods Cell lysates prepared from 293FT cells were separated using gel filtration chromatography .Then, the eluate fractions containing γTuRC or γ-tubulin small complexes (γTuSC ) were determined by immunoblotting .Results As the constitutive components of γTuRC,γ-tubulin,γ-tubulin complex protein 2 (GCP2), GCP3 and GCP4 were eluted and enriched in the fourth fraction .The molecular mass of eluates in the fourth fraction was about 2000 ×10 3 .Following γTuRC, the constitutive components ofγTuSC including γ-tubulin, GCP2 and GCP3 were eluted and enriched in the fourteenth fraction .The molecular mass of eluates in the fourteenth fraction was about 310 ×10 3 .Unassembled free components were washed out in the eighteenth and subsequent fractions .γTuRC could be detected in the corresponding fractions by negative-PAGE separation .ConclusionγTuRC and γTuSC were successfully separated from the unassembled free components in the fourth ( 4#) and fourteenth (14#) eluted fraction, respectively.The eluates containing ofγTuRC orγTuSC can be used for microtubule assembly research.