Efficient Production of Recombinant Membrane Proteins (459-501)

在这一部分中，我们将开发增强膜蛋白和膜蛋白复合体的方法 在原核和真核表达系统中均有表达。对于每个目标，我们将使用技术来 突变靶序列本身，否则我们将突变产生每个靶的宿主细胞。 最初，我们将对宿主细胞进行化学诱变，并使用容易出错的扩增来诱变靶标。 改善表情的选择将基于两个不同的屏幕，一个使用绿色荧光的视觉 蛋白质(GFP)，以及一种基于抗生素耐药性选择的方法。 对于蛋白质复合体，我们将利用带有内部核糖体进入位点(IRES)的载体进行共表达 与光谱上不同的标记(如GFP或YFP)相关联的每个目标的 用于选择。使用光谱不同的标记允许选择高表达ALL的细胞 建筑群的组成部分。最后，我们将使用直接连接到不同荧光团的目标进行跟踪 以及使用层析技术选择稳定的络合物。

In this section, we will develop methods to enhance membrane protein and membrane protein complex production in both prokaryotic and eukaryotic expression systems. For each target, we will use techniques to mutagenize the target sequence itself, or we will mutagenize the host cells in which each target is produced. Initially, we will use chemical mutagenesis for host cells, and error-prone amplification to mutagenize targets. Selection for improved expression will be based on two different screens, one visual using green fluorescent protein (GFP), and one based on selection through antibiotic resistance. For protein complexes, we will make use of vectors with internal ribosome entry sites (IRES) for the coexpression of each target associated with a spectrally distinct marker (such as GFP or YFP), which will be used for selection. The use of spectrally distinct markers allows for the selection of cells highly expressing all components of a complex. Finally, we will use targets directly attached to different fluorophores for tracking and selection of stable complexes using chromatographic techniques.