菌体显示酵母生物酶基质的演化和应用到细胞表面蛋白质的双色量子点标记
Irwin Chen1, Yoon-Aa Choi, Alice Y Ting
1Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Journal of the American Chemical Society
|May 3, 2007
概括
研究人员使用酵母生物酶 (yBL) 和一种新型酵母受体 (yAP) 开发了一种新的直角蛋白质标签系统. 这使得同一个细胞中协同表达的蛋白质的双色量子点成像成为可能.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 化学生物学 化学生物学
背景情况:
- 特定地点的蛋白质标签对于生物研究至关重要.
- 大肠杆菌生物酶 (BirA) 和其受体 (AP) 能够对重组蛋白进行标记.
- 现有的方法缺乏对多种蛋白质同时进行标记的正交性.
研究的目的:
- 为了设计一个新的,直角的生物素连接酶-受体 (AP) 对,用于特定位点的蛋白质标签.
- 为了使同一个细胞内的共同表达的蛋白质能够采用双色图像.
主要方法:
- 用菌体显示来选择一种新的15-氨基酸基质 (酵母受体,yaP) 用于酵母生物酶 (yBL).
- 通过证明yBL不识别原始AP,BirA不识别yAP,从而证实了正交性.
- yAP与各种重组蛋白质融合,并由yBL. yBL特定地生物化.
主要成果:
- 一个新的直角生物素酶-AP对 (yBL-yAP) 已成功设计.
- yBL-yAP在N端,C端和内部部位特别标记的融合蛋白.
- 证明了AP和yAP融合蛋白与明显的量子点的同表达和差异标记.
- yBL显示出高序列特异性,避免了内源细胞蛋白的生物化.
结论:
- yBL-yAP系统为正交的,特定位置的蛋白质标签提供了一个新的工具.
- 这种方法促进了两色成像,并扩大了在酵母和哺乳动物细胞系统中的应用.
- 该工程系统为蛋白质标签技术提供了增强的特异性和多功能性.
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