An efficient screening method for purifying and crystallizing membrane proteins using modified clear -native PAGE

作者：Suzuki Nanao; Takamuku Yuuki; Asakawa Tomohiro; Inai Makoto; Hino Tomoya; Iwata So; Kan Toshiyuki; Murata Takeshi^*

来源：Analytical Biochemistry, 2018, 548: 7-14.

DOI：10.1016/j.ab.2018.02.007

• Clear-native PAGE
• Membrane proteins
• Purification
• Crystallization
• G protein-coupled receptor
• Stability

Membrane proteins, such as G-protein coupled receptors, control communication between cells and their environments and are indispensable for many cellular functions. Nevertheless, structural studies on membrane proteins lag behind those on water-soluble proteins, due to their low structural stability, making it difficult to obtain crystals for X-ray crystallography. Optimizing conditions to improve the stability of membrane proteins is essential for successful crystallization. However, the optimization usually requires large amounts of purified samples, and it is a time-consuming' and trial-and-error process. Here, we report a rapid "method for pre crystallization screening of membrane proteins using Clear Native polyacrylamide gel electrophoresis (CN-PAGE) with the modified Coomassie Brilliant Blue G-250 (mCBB) stain that was reduced in sodium formate. Ay, adenosine receptor (A(2A)AR) was selected as a target membrane protein, for which we previously obtained the,"crystal structure using an antibody, and was expressed as a red fluorescent protein fusion for in-gel fluorescence detection. The mCBB CN-PAGE method enabled the optimization of the solubilization, purification, and crystallization conditions of A(2A)AR using the solubilized membrane fraction expressing the protein without purification procedures. These data suggest the applicability of mCBB CN-PAGE technique to a wide variety of integral membrane proteins.

• 出版日期2018-5-1