Efficient Production of Recombinant Membrane Proteins (459-501)

在这一节中，我们将开发方法，以提高膜蛋白和膜蛋白复合物 在原核和真核表达系统中的生产。对于每个目标，我们将使用技术来 诱变靶序列本身，或者我们将诱变产生每个靶的宿主细胞。 最初，我们将使用化学诱变宿主细胞，和易错扩增诱变目标。 将基于两个不同的屏幕选择改良表达，一个屏幕使用绿色荧光 一种是基于绿色荧光蛋白（GFP），另一种是基于抗生素抗性的选择。 对于蛋白质复合物，我们将使用具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物（如GFP或YFP）相关的每个靶点， 用于选择。光谱上不同的标记物的使用允许选择高度表达所有细胞的细胞。 一个复杂的组成部分。最后，我们将使用直接附着在不同荧光团上的目标进行跟踪 和使用色谱技术选择稳定的复合物。

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.