多次循环离子交换色谱纯化Cry1Ab蛋白及其肽质量谱图的分析

Purification of Cry1Ab protein by multi-cycles ion-exchange chromatography and its peptide mass mapping

采用Q-Sepharose FF离子交换柱对初步纯化的Cry1Ab杀虫蛋白样品进行了多次循环精细纯化,洗脱模式为离子强度台阶梯度,流动相为Tris-HCl缓冲液加NaCl,盐浓度从0.1 mol.L-1变化到1.0 mol.L-1.结果表明,Cry1Ab蛋白在NaCl浓度为0.4 mol.L-1时从柱上被洗脱,并与杂蛋白很好地分离.色谱分离机制符合顶替色谱机制.经3次循环分离后可获得高纯度的Cry1Ab蛋白.用SDS-PAGE分析了蛋白的纯度及表观分子量,电泳胶上分离的蛋白进行原位酶切后,用基质辅助激光解析-飞行时间质谱对纯化蛋白进行肽质量谱的分析,Mascot数据库搜索结果确证了该纯化蛋白酶解肽段80%符合已知的Cry1Ab蛋白特征肽段.

The coarse purified CrylAb toxin was further purified on Q-Sepharose FF ion-exchange column by multicycles separation method. Ion strength of the mobile phase increased stepwise in the elution process, in which the concentration of NaCI increased from 0. 1 mol·L^-1 to 1.0 mol·L^-1 dissolved in Tris HCI buffer. Results showed that CrylAb protein could be eluted when the concentration of NaCl reached 0.4 mol·L^-1. and the chromatographic mechanism might be in accordance with the molecular replacing mechanism. The highly purified Cry1Ab protein could be obtained after the protein sample was separated by three cycles of the chromatographic process. The purity and apparent molecular weight of the protein were determined by SDS-PAGE. The protein band on the gel was in-situ digested by trypsin, and the peptide mass mapping was analyzed by MALDI-TOF. The resulting peptides mass was searched on Mascot database, and 80G coverage with known Cry1Ab protein was found.