Efficient segmental isotope labeling of multi-domain proteins using Sortase A
Efficient segmental isotope labeling of multi-domain proteins using Sortase A
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DOI：
10.1007/s10858-015-9981-0
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发表时间：
2015-09-01
影响因子：
2.7
通讯作者：
Sattler, Michael
中科院分区：
文献类型：
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作者：
Freiburger, Lee;Sonntag, Miriam;Sattler, Michael
多结构域蛋白质复合体的核磁共振研究为了解它们在溶液中的分子相互作用和动力学提供了独特的见解。对于大的蛋白质，区域选择性同位素标记是减少信号重叠的理想方法，但现有的方法需要广泛的优化，而且往往得不到很低的连接产率。我们提出了一种使用金黄色葡萄球菌转肽酶A对多结构域蛋白质进行分段标记的优化策略。与现有方案相比，关键的改进是(1)通过离心过滤有效地去除切割的多肽片段，(2)设计了可切割和不可切割的亲和标签用于纯化。我们的方法可以常规生产毫克量的纯化片段标记蛋白，用于核磁共振和其他生物物理研究。
NMR studies of multi-domain protein complexes provide unique insight into their molecular interactions and dynamics in solution. For large proteins domain-selective isotope labeling is desired to reduce signal overlap, but available methods require extensive optimization and often give poor ligation yields. We present an optimized strategy for segmental labeling of multi-domain proteins using the S. aureus transpeptidase Sortase A. Critical improvements compared to existing protocols are (1) the efficient removal of cleaved peptide fragments by centrifugal filtration and (2) a strategic design of cleavable and non-cleavable affinity tags for purification. Our approach enables routine production of milligram amounts of purified segmentally labeled protein for NMR and other biophysical studies.