Novartis Institutes for BioMedical Research, Basel, Switzerland.
J Biomol NMR. 2011 Dec;51(4):449-56. doi: 10.1007/s10858-011-9570-9. Epub 2011 Oct 2.
An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract, similar protein amounts as with full media are obtained, without compromising on isotope incorporation. In single and dual amino acid labeling experiments incorporation ratios are consistently ≥90% for all amino acids tested. This enables NMR studies of eukaryotic proteins and their interactions even for proteins with low expression levels. We show applications with human kinases, where protein-ligand interactions are characterized by 2D [(15)N, (1)H]- and [(13)C, (1)H]-HSQC spectra.
本文介绍了一种用于昆虫细胞中氨基酸类型选择性同位素标记的简便、强大的方法。它依赖于廉价的商业培养基，并且可以在没有复杂基础设施的实验室中实施。与以前的方案不同，以前的方案要么获得高蛋白质含量，要么获得高掺入率，而在这里我们同时实现了这两个目标。通过向培养基中补充经过精心考虑的酵母提取物的量，可以获得与完全培养基相同的蛋白质含量，而不会影响同位素掺入。在单氨基酸和双氨基酸标记实验中，所有测试的氨基酸的掺入率始终≥90%。这使得即使对于表达水平低的蛋白质，也可以进行 NMR 研究真核蛋白质及其相互作用。我们展示了与人激酶的应用，其中通过 2D [(15)N，(1)H]-和 [(13)C，(1)H]-HSQC 谱来表征蛋白质-配体相互作用。
