【摘要】 目的: 制备有高生物活性的重组蓖麻毒素A链蛋白(RTA)。方法: 在RTA序列的C末端引入KDEL信号肽,克隆到含有硫氧还蛋白(Trx)的融合表达载体pET32a中, 转入大肠杆菌BL21, 在低温 ( 20℃)下用低浓度 ( 0. 4mmol/L)IPTG诱导重组质粒进行表达。表达上清用钴离子亲和层析柱进行纯化, 20~100mmol/L咪唑溶液洗脱目的蛋白。纯化蛋白经SDS- PAGE电泳与Westernblot鉴定后, 对pUC19质粒进行超螺旋dsDNA裂解研究。结果: 每升细菌培养物回收约60mg的纯化蛋白, 纯度大于 90%, Mr约 45 000, 且 3μg纯化蛋白即可以对 1μg超螺旋dsDNA产生明显的裂解活性。结论: 利用pET32a表达系统可以快速获得大量有高生物活性的可溶性RTA- Trx融合蛋白。更多还原

【Abstract】 AIM: To prepare recombinant ricin A-chain(RTA) protein with high biological activity. METHODS: RTA gene containing KDEL sequence at the carboxyl terminal was cloned in pET32a vector, which was fused with thioredoxin. Furthermore, the constructed recombinant plasmid was transformed into the competent cell BL21, and induced with low concentration of IPTG (0.4 mmol/L) under low temperature (20℃). After binding to Co 2+ chelating column, the expressed supernatants were eluted by applying imidazole solutions with the concentration from 20 to 100 mmol/L. The purified protein was identified with SDS-PAGE and Western blot analysis and was used to cleave supercoiled dsDNA. RESULTS: About 60 mg fusion proteins were obtained from 1 000 mL cultures, with purity above 90% and M r 45 000. The cleavage of supercoiled dsDNA demonstrated that RTA-Trx fusion proteins could significantly cleave supercoiled dsDNA as native RTA. CONCLUSION: The pET32a vector expression system can be used to produce a mass of soluble RTA-Trx fusion proteins with high biological activity.更多还原