Assessment of membrane protein expression and stability using a split green fluorescent protein reporter

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• 作者：Arturo Rodrí ; guez-Banqueri^a ; ¹ ; Lukasz Kowalczyk^a ; ¹ ; Manuel Palací ; n^a ; ^b ; ^c ; José ; Luis Vá ; zquez-Ibar^a ; ^d ; ^{jl.vazquez.ibar@gmail.com}
• 关键词：Membrane protein ; Protein crystallography ; GFP ; SteT
• 刊名：Analytical Biochemistry
• 出版年：2012
• 期刊代码：93_00032697
• 类别：imm
• 出版时间：1 April, 2012
• 卷：423
• 期：1
• 页码：7-14
• 文件大小：707 K

摘要

Membrane proteins are challenging targets for structural biologists. Finding optimal candidates for such studies requires extensive and laborious screening of protein expression and/or stability in detergent. The use of green fluorescent protein (GFP) as a reporter has enormously facilitated these studies; however, its 238 residues can potentially alter the intrinsic properties of the target (e.g., expression or stability). With the aim of minimizing undesired effects of full-length GFP, here we describe the utility of a split GFP reporter during precrystallization studies of membrane proteins. GFP fluorescence appeared by complementation of the first 15 residues of GFP (GFP₁₁) (fused to the C terminus of a membrane protein target) with the remaining nonfluorescent GFP (GFP_1-10). The signal obtained after sequential expression of SteT (l-serine/l-threonine exchanger of Bacillus subtilis) fused to GFP₁₁ followed by GFP_1-10 specifically measured the protein fraction inserted into the Escherichia coli cytoplasmic membrane, thereby discarding protein aggregates confined as inclusion bodies. Furthermore, in vitro complementation of purified SteT-GFP₁₁ with purified GFP_1-10 was exploited to rapidly assess the stability of wild-type and G294V mutant versions of SteT-GFP₁₁ following detergent solubilization and purification. This method can be applied in a medium- to high-throughput manner with multiple samples.