Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells
Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells
复制标题
DOI：
10.1038/nprot.2011.453
复制
发表时间：
2012-03-01
期刊：
影响因子：
14.8
通讯作者：
Stroud, Robert M.
中科院分区：
文献类型：
--
作者：
Chaudhary, Sarika;Pak, John E.;Stroud, Robert M.
人膜蛋白的X射线晶体结构虽然具有极大的潜在影响，但与原核膜蛋白相比，X射线晶体结构的代表性严重不足。这其中的一个关键原因是，人膜蛋白可能很难在适合结构研究的水平和质量上表达。该方案描述了我们从缺乏N-乙酰氨基葡萄糖基转移酶I(GNTI(-))的克隆人胚胎肾293(HEK293S)细胞中过表达人膜蛋白的方法，并最近用于我们的2.1埃X射线晶体结构测定。在鉴定出高表达的细胞系后，悬浮细胞培养物以一种简单的方式放大，要么使用旋转烧瓶，要么使用纤维袋生物反应器，最终纯化产量接近每升培养基膜蛋白0.5毫克。这里描述的方案是可靠和经济有效的，可以用来表达本来对哺乳动物细胞有毒的蛋白质，并且可以在8-10周内完成。
X-ray crystal structures of human membrane proteins, although potentially of extremely great impact, are highly underrepresented relative to those of prokaryotic membrane proteins. One key reason for this is that human membrane proteins can be difficult to express at a level, and at a quality, suitable for structural studies. This protocol describes the methods that we use to overexpress human membrane proteins from clonal human embryonic kidney 293 ( HEK293S) cells lacking N-acetylglucosaminyltransferase I (GnTI(-)), and was recently used in our 2.1-angstrom X-ray crystal structure determination of human RhCG. Upon identification of highly expressing cell lines, suspension cell cultures are scaled up in a facile manner either using spinner flasks or cellbag bioreactors, resulting in a final purified yield of similar to 0.5 mg of membrane protein per liter of medium. The protocol described here is reliable and cost effective, can be used to express proteins that would otherwise be toxic to mammalian cells and can be completed in 8-10 weeks.