Efficient Production of Recombinant Membrane Proteins (459-501)

重组膜蛋白的高效生产 (459-501)

基本信息

批准号：
9050045
负责人：
WAYNE A. HENDRICKSON
金额：
$ 21.18万
依托单位：
NEW YORK STRUCTURAL BIOLOGY CENTER
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-01 至 2021-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9050045
关键词：
Amino Acids Antibiotic Resistance Antibiotics Cell Line Cells Chemicals Complex Derivation procedure Elements Enzymes Escherichia coli Escherichia coli Proteins Eukaryotic Cell Fluorescence Gene Targeting Genes Green Fluorescent Proteins Individual Integral Membrane Protein Internal Ribosome Entry Site Lead Libraries Lipids Measurement Membrane Membrane Proteins Methods Molecular Chaperones Mutagenesis Mutation Plasmids Population Post-Translational Protein Processing Process Production Proteins Recombinants Reporting Resistance Ribosomes Saccharomyces cerevisiae Screening procedure Selection Criteria Source Structure System Techniques Toxic effect Transcript Variant Visual base cell transformation expression vector fluorophore genetic selection improved in vivo inducible gene expression interest member mutant protein complex research study vector

项目摘要

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.

在本节中，我们将开发增强膜蛋白和膜蛋白复合物的方法在原核和真核表达系统中生产。对于每个目标，我们将使用技术来诱变目标序列本身，或者我们将诱变产生每个目标的宿主细胞。最初，我们将对宿主细胞使用化学诱变，并使用易错扩增来诱变靶标。改进表达的选择将基于两种不同的屏幕，一种使用绿色荧光的视觉效果蛋白质（GFP），以及一种基于抗生素抗性选择的蛋白质。对于蛋白质复合物，我们将利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（例如 GFP 或 YFP）相关的每个目标，这将是用于选择。使用光谱上不同的标记可以选择高度表达所有复合体的组成部分。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以及使用色谱技术选择稳定的复合物。