Freiburger Lee, Sonntag Miriam, Hennig Janosch, Li Jian, Zou Peijian, Sattler Michael
Institute of Structural Biology, Helmholtz Zentrum München, 85764, Neuherberg, Germany.
Center for Integrated Protein Science Munich (CIPSM) at Department of Chemistry, Technische Universität München, Lichtenbergstr.4, 85747, Garching, Germany.
J Biomol NMR. 2015 Sep;63(1):1-8. doi: 10.1007/s10858-015-9981-0. Epub 2015 Aug 30.
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.
多结构域蛋白质复合物的核磁共振（NMR）研究为其在溶液中的分子相互作用和动力学提供了独特的见解。对于大型蛋白质，需要进行结构域选择性同位素标记以减少信号重叠，但现有方法需要大量优化，且连接产率往往较低。我们提出了一种使用金黄色葡萄球菌转肽酶分选酶A对多结构域蛋白质进行片段标记的优化策略。与现有方案相比，关键改进之处在于：（1）通过离心过滤有效去除切割后的肽片段；（2）设计可切割和不可切割的亲和标签用于纯化。我们的方法能够常规生产毫克量的纯化片段标记蛋白质，用于NMR和其他生物物理研究。
