Tools for Exceptional Overexpression and Structural Stabilization of Membrane Proteins in Mammalian Cells

在哺乳动物细胞中实现膜蛋白异常过度表达和结构稳定的工具

• 批准号: 9199227
• 负责人: Arne Gennerich
• 金额: $ 32.29万
• 依托单位: LUCIGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9199227
• 关键词: Adrenergic Receptor; Alpha Cell; Cell Culture System; Cell Culture Techniques; Cells; Chimeric Proteins; Crystallization; Data; Detection; Detergents; Drug Design; Escherichia coli; Exhibits; Fluorescence; G-Protein-Coupled Receptors; Generations; Goals; High temperature of physical object; Human; Individual; Insecta; Integral Membrane Protein; Mammalian Cell; Measures; Membrane Proteins; Methods; Pattern; Peptides; Phase; Post-Translational Protein Processing; Process; Production; Proteins; Recombinant Proteins; Recombinants; Solubility; Structure; System; Technology; Time; Variant; Viral; Work; Yeasts; base; cost; expression vector; glycosylation; human disease; improved; novel; overexpression; protein expression; protein purification; public health relevance; receptor; screening; structural biology; therapeutic development; thermostability; tool

 DESCRIPTION (provided by applicant): In this Phase I proposal, we plan to develop two technologies to dramatically increase the expression and crystallization of properly folded recombinant membrane proteins from mammalian cell culture. The most common methods to produce high yields of recombinant proteins at low cost rely on bacterial, yeast, or insect-cell expression. However, the proteins produced in these systems are very difficult to purify in native form, and they lack the mammalian glycosylation patterns that are often necessary for proper folding and native activity. We plan to develop a novel protein fusion partner that enables mammalian expression of proteins that are otherwise nearly impossible to produce. A second technology will add an internal motif to greatly enhance structural stability and facilitate crystallization of the target membrane protein. Together, these technologies have the potential to improve protein yields in mammalian cell culture by over 20-fold and to allow crystallization of proteins that are currently impossible to study.

 描述（由申请人提供）：在该第一阶段提案中，我们计划开发两种技术来显着提高哺乳动物细胞培养物中正确折叠的重组膜蛋白的表达和结晶，这是以低产量生产重组蛋白的最常见方法。然而，这些系统中产生的蛋白质很难以天然形式纯化，并且它们缺乏正确折叠和形成所需的哺乳动物糖基化模式。我们计划开发一种新的蛋白质融合伙伴，使哺乳动物能够表达几乎不可能产生的蛋白质，第二种技术将添加一个内部基序，以大大增强结构稳定性并促进目标膜蛋白的结晶。这些技术有可能将哺乳动物细胞培养物中的蛋白质产量提高 20 倍以上，并允许结晶 目前无法研究的蛋白质。