使用阿斯巴拉基尼尔联酶进行pH控制的蛋白质正交联
Dingpeng Zhang1, Zhen Wang1, Side Hu1
1School of Biological Sciences, Nanyang Technological University, Singapore 637551.
Journal of the American Chemical Society
|June 7, 2021
概括
类基酶 (PAL) 现在可以在酸性pH条件下有效地在基结处结合. 这一突破使得顺序,pH控制的结合能够产生复杂的,多功能蛋白质生物.
科学领域:
- 生物化学
- 蛋白质工程
- 化学生物学
背景情况:
- 已知类基酶 (PALs) 在类基 (Asn) 残留物上具有高效的结.
- PAL通常需要中性pH用于Asn结合和酸性pH用于aspartyl (Asp) 结合,这限制了Asp特定的结合效率.
- 由于在酸性pH下所感知的效率低下,PAL用于Asp特异性结合尚未得到充分研究.
研究的目的:
- 研究和优化PAL酶的使用以实现高效的Asp特异性结.
- 开发一个pH控制的直角结合策略,使用PAL进行序列标记.
- 展示这种方法在制造双标记蛋白质生物制剂中的应用.
主要方法:
- 在酸性pH下对P1-Asp基质进行PAL酶,包括VyPAL2的催化活性评估 (4-5).
- 将PALs的活性与Asp结合的Sortase A进行比较.
- 开发并应用了对蛋白质 (sfGFP和affibody) 进行双重标记的顺序,pH控制的结合策略.
主要成果:
- 与类酶A相比,PAL酶在pH4-5时对P1-Asp基质具有显著更高的催化活性.
- 在酸性条件下使用PAL在Asp结处实现有效的分子内和分子间结合.
- 成功地对具有不同的有效载荷 (,药物,染料) 的蛋白质进行了N-to-C和C-to-N双标签.
结论:
- 在酸性pH下,PAL酶对Asp特异性结合具有实用性,克服了先前的限制.
- 使用PALs进行pH控制的正交联结使蛋白质的精确序列修饰成为可能.
- 这种方法有助于制备复杂的多功能蛋白质生物制剂,用于药理应用.
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