Vandecaetsbeek Ilse, Vangheluwe Peter
Department of Cellular and Molecular Medicine, Laboratory of Cellular Transport Systems, KU Leuven, Herestraat 49, 802, B3000, Leuven, Belgium.
Methods Mol Biol. 2016;1377:79-85. doi: 10.1007/978-1-4939-3179-8_9.
Membrane protein purification often yields rather unstable proteins impeding functional and structural protein characterization. Low protein stability also leads to low purification yields as a result of protein degradation, aggregation, precipitation, and folding instability. It is often required to optimize buffer conditions through numerous iterations of trial and error to improve the homogeneity, stability, and solubility of the protein sample demanding high amounts of purified protein. Therefore we have set up a fast, simple, and high-throughput time-dependent thermostability-based assay at low protein cost to identify protein stabilizing factors to facilitate the handling and characterization of membrane proteins by subsequent structural and functional studies.
膜蛋白纯化常常会产生相当不稳定的蛋白质,这阻碍了对蛋白质功能和结构的表征。由于蛋白质降解、聚集、沉淀以及折叠不稳定性,低蛋白质稳定性还会导致低纯化产率。通常需要通过大量反复试验来优化缓冲条件,以提高蛋白质样品的均一性、稳定性和溶解性,而这需要大量纯化蛋白质。因此,我们建立了一种快速、简单且高通量的基于时间依赖性热稳定性的检测方法,成本低廉,用于鉴定蛋白质稳定因子,以便通过后续的结构和功能研究促进膜蛋白的处理和表征。