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sec途径的蛋白质转运模式。然而，由于各种原因，许多重组蛋白与通过sec途径的分泌不相容。 主要研究者（PI）提出了通过利用达特分泌途径的显著特性来克服细菌中分泌蛋白表达的局限性的研究。 PI建议利用达特途径的独特功能来克服与分泌蛋白生产相关的限制，并开发用于组合蛋白筛选和分析的全新技术平台。 建议：（1）开发用于通过达特途径高水平表达分泌蛋白的有用技术，具体地，PI将：（i）采用遗传手段来鉴定使通过达特途径输出的蛋白的通量最大化的前导肽;（ii）利用细胞工程方法进一步优化蛋白质分泌和（iii）详细检查生长条件对分泌性蛋白质生产的影响，（2）开发利用达特途径展示蛋白质文库的方法，然后研究改变蛋白质分泌途径将导致分离能够执行所需功能的额外和/或新蛋白质的假设，和（3）开发利用达特途径检测类似于酵母双杂交系统的蛋白质-蛋白质相互作用的新技术。

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.