Blakeley Brett D, Chapman Alex M, McNaughton Brian R
Department of Chemistry, Colorado State University, Fort Collins, Colorado, USA.
Mol Biosyst. 2012 Aug;8(8):2036-40. doi: 10.1039/c2mb25130b. Epub 2012 Jun 12.
Split-GFP reassembly is an operationally simple in vivo technique used to identify and study interactions involving proteins and/or peptides. However, the instability of split-GFP fragments and their susceptibility to aggregation place limitations on the broader use of split-GFP reassembly. Supercharged proteins, including supercharged GFP, are variants with high theoretical negative or positive charge that are resistant to aggregation. We show that a split-superpositive GFP (split-spGFP) variant reassembles faster and more efficiently than previously reported split-sg100 GFP and split-folding-reporter GFP (split-frGFP) systems. In addition, interaction-dependent split-spGFP reassembly is efficient at physiological temperature. The increased efficiency and robustness of split-spGFP reassembly make this reporter system ideal for identifying and studying interactions involving proteins and/or peptides in vivo, and may be particularly useful for identifying or studying interactions involving proteins or peptides that are themselves susceptible to aggregation.
分裂型绿色荧光蛋白（Split-GFP）重组装是一种操作简单的体内技术，用于识别和研究涉及蛋白质和/或肽的相互作用。然而，分裂型绿色荧光蛋白片段的不稳定性及其聚集倾向限制了分裂型绿色荧光蛋白重组装技术的广泛应用。带电荷增强的蛋白质，包括带电荷增强的绿色荧光蛋白，是具有高理论负电荷或正电荷且抗聚集的变体。我们发现，一种分裂型超正电荷绿色荧光蛋白（split-spGFP）变体的重组装速度比先前报道的分裂型sg100绿色荧光蛋白和分裂型折叠报告绿色荧光蛋白（split-frGFP）系统更快、更高效。此外，依赖于相互作用的分裂型spGFP重组装在生理温度下也很高效。分裂型spGFP重组装效率和稳定性的提高，使得该报告系统成为体内识别和研究涉及蛋白质和/或肽的相互作用的理想选择，对于识别或研究本身易于聚集的蛋白质或肽之间的相互作用可能特别有用。
