Experimental Physics: Genetic Biophysics, Freie Universität Berlin, Berlin, Germany.
Methods Mol Biol. 2021;2191:29-48. doi: 10.1007/978-1-0716-0830-2_3.
For a successful characterization of channelrhodopsins with biophysical methods like FTIR, Raman, EPR and NMR spectroscopy and X-ray crystallography, large amounts of purified protein are requested. For proteins of eukaryotic origin, which are poorly expressing in bacterial systems or not at all, the yeast Pichia pastoris represents a promising alternative for overexpression. Here we describe the methods for cloning, overexpression and mutagenesis as well as the purification procedures for channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) and the scaffold protein MSP1D1 for reconstitution of the membrane proteins into nanodiscs. Finally, protocols are provided to study CaChR1 by FTIR difference spectroscopy and by time-resolved UV/Vis spectroscopy.
为了成功地使用生物物理方法（如傅里叶变换红外光谱（FTIR）、拉曼光谱、电子顺磁共振（EPR）和核磁共振（NMR）光谱以及 X 射线晶体学）对通道蛋白进行表征，需要大量纯化的蛋白质。对于在细菌系统中表达不佳或根本不表达的真核来源的蛋白质，毕赤酵母（Pichia pastoris）是过表达的一种很有前途的替代方法。在这里，我们描述了从莱茵衣藻（Chlamydomonas reinhardtii）（CrChR2）克隆、过表达和突变以及从 Chlamydomonas augustae（CaChR1）纯化通道蛋白-2和支架蛋白 MSP1D1 以及将膜蛋白重构成纳米盘的方法。最后，还提供了通过傅里叶变换红外光谱（FTIR）差谱法和时间分辨紫外/可见光谱法研究 CaChR1 的方案。
