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

 描述（由申请人提供）：在本I期提案中，我们计划开发两种技术，以显著增加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。以低成本生产高产量重组蛋白的最常见方法依赖于细菌、酵母或昆虫细胞表达。然而，在这些系统中产生的蛋白质很难以天然形式纯化，并且它们缺乏通常对于适当折叠和天然活性所必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伴侣，使哺乳动物表达的蛋白质，否则几乎不可能产生。第二种技术将添加一个内部基序，以大大提高结构稳定性，并促进靶膜蛋白的结晶。总之，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，并允许结晶化。 目前无法研究的蛋白质。

 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.