在没有先前净化的情况下,对蛋白质进行化学酶可逆固定和标记
Mohammad Rashidian1, James M Song, Rachel E Pricer
1Department of Chemistry, University of Minnesota, Minneapolis, 55454, United States.
Journal of the American Chemical Society
|March 23, 2012
概括
这项研究引入了一种新的方法,用于使用含有的基质与蛋白质法纳西尔转移酶 (PFTase) 进行特定位点的蛋白质修饰. 这使得可用于生物技术和治疗应用的蛋白质的多功能标签和结合,甚至在原始提取物中.
科学领域:
- 生物化学 生物化学
- 化学生物学 化学生物学
- 生物技术是生物技术.
背景情况:
- 特定位置的蛋白质修饰对于生物和生物技术应用至关重要.
- 蛋白法纳西转移酶 (PFTase) 具有高特异性,可用于修改具有生物直角功能的蛋白质,如亚化物和化物.
- 使用PFTase和特定基质,可以生成化功能化蛋白质.
研究的目的:
- 为了评估含有的分子作为PFTase的基质.
- 为了证明PFTase介导的阿尔德海德合并对随后的生物对等结合的实用性.
- 开发一种通用方法,用于在原始细胞提取物中对特定区域的蛋白质进行标记和修改.
主要方法:
- 绿色光蛋白 (GFP) 与含有的基质的酶性修饰使用PFTase.
- 在蛋白质固定,光标记和PEGylation中形成oxime和hydrazone.
- 通过 transoximization 的可逆固定.
- 从原始大肠杆菌提取物中选择性标记和净化GFP.
- 葡萄糖依赖型胰岛素型多 (GIP) 的PEGylation.
主要成果:
- 通过使用PFTase.成功修改纯化GFP与 aldehyde功能.
- 通过氧化物/化化学反应,对化物修饰的蛋白质进行了有效的标记,固定和PEGylated.
- 实现了可逆固定和释放修改后的GFP.
- 在原始大肠杆菌提取物中进行了特定地点的标签和GFP的修改,随后进行了净化.
- 生产了PEGylated GIP,突出了治疗潜力.
结论:
- PFTase可以利用含有的基质进行特定位点的蛋白质修饰.
- 这种方法可以实现蛋白质的多功能下游结合 (标记,固定,PEGylation).
- 这种方法在原始细胞提取物中是有效的,消除了先前蛋白质净化的需要.
- 这一战略为大规模生产用于治疗和工业用途的蛋白质合物提供了巨大的潜力.
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