A Novel Technology Platform for Protein Engineering and Expression

许多与分泌蛋白相关的生物技术应用几乎完全依赖于蛋白质运输的sec途径模式。然而，由于各种原因，许多重组蛋白与sec途径的分泌不相容。首席研究员（PI）建议通过利用Tat分泌途径的显著特性来克服细菌分泌蛋白表达的局限性。PI建议利用Tat通路的独特特征来克服与分泌蛋白产生相关的限制，并开发用于组合蛋白筛选和分析的全新技术平台。建议：(1)开发一种有用的技术，用于通过Tat途径高水平表达分泌蛋白，特别是PI将：(i)采用遗传方法鉴定先导肽，使通过Tat途径输出的蛋白质通量最大化；（ii）利用细胞工程方法进一步优化蛋白质分泌；（iii）详细检查生长条件对分泌蛋白产生的影响；(2)利用Tat途径开发蛋白质文库的展示方法，然后研究改变蛋白质分泌途径将导致分离出能够执行所需功能的额外和/或新蛋白质的假设。(3)利用Tat通路开发类似酵母双杂交系统的蛋白-蛋白相互作用检测新技术。

Many biotechnology applications related to secreted proteins have relied almost exclusively on the sec pathway mode of protein transport. However, many recombinant proteins are incompatible with secretion via the sec pathway for various reasons. The Principal Investigator (PI) proposes research to overcome the limitations of secretory protein expression in bacteria by capitalizing on the remarkable properties of the Tat secretion pathway. The PI proposes to exploit the unique features of the Tat pathway to overcome the limitations associated with secretory protein production and to develop completely new technology platforms for combinatorial protein screening and analysis. It is proposed to: (1) develop a useful technology for the high level expression of secretory proteins via the Tat pathway, specifically the PI will: (i) employ genetic means to identify leader peptides that maximize the flux of proteins exported though the Tat pathway; (ii) utilize cell engineering methods to further optimize protein secretion and (iii) examine in detail the effect of growth conditions on secretory protein production, (2) develop methods for the display of protein libraries by utilizing the Tat pathway and then investigate the hypothesis that altering the pathway of protein secretion will result in the isolation of additional and/or novel proteins capable of performing a desired function, and (3) develop a new technology for the detection of protein-protein interactions analogous to the yeast two-hybrid system by exploiting the Tat pathway.