Efficient Production of Recombinant Membrane Proteins (459-501)

重组膜蛋白的高效生产 (459-501)

• 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
• 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

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