Marley J, Lu M, Bracken C
Department of Biochemistry, Weill Medical College of Cornell University, New York, NY 10021, USA.
J Biomol NMR. 2001 May;20(1):71-5. doi: 10.1023/a:1011254402785.
A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for 13C labeling of the C-terminal domain of angiopoietin-2, 15N labeling of ubiquitin and for 2H/13C/15N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.
本文介绍了一种快速高效制备同位素标记重组蛋白的方法。该方法已在血管生成素-2 C端结构域的13C标记、泛素的15N标记以及大肠杆菌外膜脂蛋白Lpp-56的2H/13C/15N标记中得到验证。该生产方法先用未标记的丰富培养基培养细胞，然后在高细胞密度下将其转移到少量标记培养基中。经过短暂的生长恢复和未标记代谢物清除后，诱导细胞。使用简单的摇瓶培养，所获得的表达产量可使同位素成本降低四至八倍。
