Plucinsky Sarah M, Root Kyle T, Glover Kerney Jebrell
Department of Chemistry, Lehigh University, 6 E. Packer Ave, Bethlehem, PA 18015, USA.
Department of Chemistry, Lock Haven University, 301 W. Church St, Lock Haven, PA 17745, USA.
Protein Expr Purif. 2018 Mar;143:34-37. doi: 10.1016/j.pep.2017.10.012. Epub 2017 Oct 21.
The purification of membrane proteins can be challenging due to their low solubility in conventional detergents and/or chaotropic solutions. The introduction of fusion systems that promote the formation of inclusion bodies has facilitated the over-expression of membrane proteins. In this protocol, we describe the use of perfluorooctanoic acid (PFOA) as an aid in the purification of highly hydrophobic membrane proteins expressed as inclusion bodies. The advantage of utilizing PFOA is threefold: first, PFOA is able to reliably solubilize inclusion bodies, second, PFOA is compatible with nickel affinity chromatography, and third, PFOA can be efficiently dialyzed away to produce a detergent free sample. To demonstrate the utility of employing PFOA, we expressed and purified a segment of the extremely hydrophobic membrane protein caveolin-1.
由于膜蛋白在传统洗涤剂和/或离液剂溶液中的溶解度较低，其纯化过程可能具有挑战性。促进包涵体形成的融合系统的引入有助于膜蛋白的过表达。在本方案中，我们描述了使用全氟辛酸（PFOA）辅助纯化以包涵体形式表达的高度疏水膜蛋白。使用PFOA的优势有三方面：第一，PFOA能够可靠地溶解包涵体；第二，PFOA与镍亲和层析兼容；第三，PFOA可以通过透析有效去除，从而得到不含洗涤剂的样品。为了证明使用PFOA的实用性，我们表达并纯化了极疏水膜蛋白小窝蛋白-1的一个片段。
