Walton Wendy J, Kasprzak Agnieszka J, Hare Joan T, Logan Timothy M
Institute of Molecular Biophysics, Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA.
J Biomol NMR. 2006 Dec;36(4):225-33. doi: 10.1007/s10858-006-9086-x. Epub 2006 Oct 25.
It is estimated that over half of all proteins are glycosylated, yet only a small number of the structures in the protein data bank are of intact glycoproteins. One of the reasons for the lack of structural information on glycoproteins is the high cost of isotopically labeling proteins expressed from eukaryotic cells such as in insect and mammalian cells. In this paper we describe modifications to commercial insect cell growth medium that reduce the cost for isotopically labeling recombinant proteins expressed from Sf9 cells. A key aspect of this work was to reduce the amount of glutamine in the cell culture medium while maintaining sufficient energy yielding metabolites for vigorous growth by supplementing with glucose and algae-derived amino acids. We present an analysis of cell growth and protein production in Sf9 insect cells expressing secreted Thy1-GFP fusion construct. We also demonstrate isotopic enrichment of the Thy-1 protein backbone with 15N and carbohydrates with 13C by NMR spectroscopy.
据估计，超过一半的蛋白质都进行了糖基化修饰，但蛋白质数据库中完整糖蛋白的结构却只有少数。糖蛋白缺乏结构信息的原因之一是对真核细胞（如昆虫和哺乳动物细胞）表达的蛋白质进行同位素标记成本高昂。在本文中，我们描述了对商业昆虫细胞生长培养基的改良，这种改良降低了对从Sf9细胞表达的重组蛋白进行同位素标记的成本。这项工作的一个关键方面是减少细胞培养基中谷氨酰胺的量，同时通过补充葡萄糖和藻类衍生的氨基酸来维持足够的能产生能量的代谢物，以实现旺盛生长。我们对表达分泌型Thy1-GFP融合构建体的Sf9昆虫细胞中的细胞生长和蛋白质生产进行了分析。我们还通过核磁共振光谱证明了Thy-1蛋白主链的15N同位素富集以及碳水化合物的13C同位素富集。
