Okar D A, Felicia N D, Gui L, Lange A J
Department of Biochemistry, School of Medicine, University of Minnesota, Minneapolis 55455, USA.
Protein Expr Purif. 1997 Oct;11(1):79-85. doi: 10.1006/prep.1997.0770.
Methods for the efficient use of the 13C-labeled nutrients, glucose and histidine, in the production of recombinant protein were developed to provide the large amount of sample required for NMR studies. The nutrient requirements were reduced by determining the minimum amount of these metabolites needed during both the growth and the induction phases of the BL21(DE3) and newly constructed BL21(DE3) histidine auxotrophic Escherichia coli cultures. These methods were developed using the separate bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase, which is expressed to high levels in the pET3a/BL21 (DE3) bacterial system. Use of the optimized expression methods reduced the requirements for the labeled nutrients, glucose and histidine, by 90 and 93.8%, respectively. The savings realized by use of the minimized media and modified induction protocols were obtained without significant reduction of the yield of purified protein. Comprehensive study of the bisphosphatase domain by NMR spectroscopy requires large amounts of protein because of its low solubility and the short lifetime (2-3 days) of the NMR samples. The significant reduction in the costs of labeled protein samples realized by the optimized expression methods can meet these sample requirements in a cost-effective way, and thereby, allow NMR studies of the bisphosphatase domain to proceed.
开发了在重组蛋白生产中高效利用13C标记营养物葡萄糖和组氨酸的方法，以提供核磁共振（NMR）研究所需的大量样品。通过确定BL21(DE3)和新构建的组氨酸营养缺陷型大肠杆菌培养物在生长和诱导阶段所需的这些代谢物的最小量，降低了营养需求。这些方法是利用大鼠肝脏6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶的单独双磷酸酶结构域开发的，该结构域在pET3a/BL21(DE3)细菌系统中高水平表达。使用优化的表达方法分别将标记营养物葡萄糖和组氨酸的需求降低了90%和93.8%。通过使用最小化培养基和改进的诱导方案实现了节约，同时纯化蛋白的产量没有显著降低。由于其低溶解度和NMR样品的短寿命（2-3天），通过NMR光谱对双磷酸酶结构域进行全面研究需要大量蛋白质。优化的表达方法显著降低了标记蛋白样品的成本，能够以具有成本效益的方式满足这些样品需求，从而使双磷酸酶结构域的NMR研究得以进行。
