Faculty of Health and Life Sciences, Coventry University, Coventry, UK.
College of Health and Life Sciences, Aston University, Birmingham, UK.
Methods Mol Biol. 2022;2507:187-199. doi: 10.1007/978-1-0716-2368-8_10.
The first crystal structures of recombinant mammalian membrane proteins were solved using high-quality protein that had been produced in yeast cells. One of these, the rat Kv1.2 voltage-gated potassium channel, was synthesized in Pichia pastoris. Since then, this yeast species has remained a consistently popular choice of host for synthesizing eukaryotic membrane proteins because it is quick, easy, and cheap to culture and is capable of posttranslational modification. Very recent structures of recombinant membrane proteins produced in P. pastoris include a series of X-ray crystallography structures of the human vitamin K epoxide reductase and a cryo-electron microscopy structure of the TMEM206 proton-activated chloride channel from pufferfish. P. pastoris has also been used to structurally and functionally characterize a range of membrane proteins including tetraspanins, aquaporins, and G protein-coupled receptors. This chapter provides an overview of the methodological approaches underpinning these successes.
使用在酵母细胞中产生的高质量蛋白质，解决了第一个重组哺乳动物膜蛋白的晶体结构。其中之一是大鼠 Kv1.2 电压门控钾通道，它是在毕赤酵母中合成的。从那时起，这种酵母仍然是合成真核膜蛋白的常用宿主，因为它易于培养，快速且廉价，并且能够进行翻译后修饰。最近在毕赤酵母中产生的重组膜蛋白的结构包括一系列人维生素 K 环氧化物还原酶的 X 射线晶体学结构，以及河豚 TMEM206 质子激活氯离子通道的冷冻电镜结构。毕赤酵母还被用于对一系列膜蛋白进行结构和功能表征，包括四跨膜蛋白、水通道蛋白和 G 蛋白偶联受体。本章概述了支持这些成功的方法学方法。
