Department of Chemistry, University of California, Riverside, California, USA.
National High Magnetic Field Laboratory and Department of Biological Science, Florida State University, Tallahassee, Florida, USA.
Nat Protoc. 2014 Apr;9(4):910-28. doi: 10.1038/nprot.2014.054. Epub 2014 Mar 20.
Naturally occurring fluorescent proteins (FPs) cloned from marine organisms often suffer from many drawbacks for cell biology applications, including poor folding efficiency at 37 °C, slow chromophore formation and obligatory quaternary structure. Many of these drawbacks can be minimized or eliminated by using protein engineering and directed evolution, resulting in superior probes for use in live-cell fluorescence microscopy. In this protocol, we provide methods for engineering a monomeric FP, for enhancing its brightness by directed evolution, and for thoroughly characterizing the optimized variant. Variations on this procedure can be used to select for many other desirable features, such as a red-shifted emission spectrum or enhanced photostability. Although the length of the procedure is dependent on the degree of optimization desired, the basic steps can be accomplished in 4-6 weeks.
从海洋生物中克隆的天然荧光蛋白 (FPs) 在用于细胞生物学应用时常常存在许多缺点，包括在 37°C 时折叠效率差、发色团形成缓慢以及必须形成四级结构。通过使用蛋白质工程和定向进化，可以最大限度地减少或消除这些缺点，从而为活细胞荧光显微镜提供更优越的探针。在本方案中，我们提供了工程化单体 FP 的方法，通过定向进化提高其亮度，并对优化的变体进行全面表征。该过程的变体可用于选择许多其他理想的特性，例如红移发射光谱或增强的光稳定性。尽管程序的长度取决于所需的优化程度，但基本步骤可以在 4-6 周内完成。
