Streptavidin engineering and characterization of putative sortases and their substrates from bacillus subtilis

该提案有两个项目。第一个项目的重点是链亲和素工程。链亲和素是一种四亚基生物素结合蛋白。由于紧密结合，链霉亲和素可作为生物素化蛋白的固定化剂。我们设计了一种新的链亲和素，命名为M6。它每个分子只有一个亚基，可以以可逆的方式结合生物素化的蛋白质。M6具有单个半胱氨酸残基，可以与携带含有半胱氨酸的生物素化肽标签CPFB的重组蛋白形成二硫键。因此，M6具有固定含有重组蛋白的CPFB标签的能力。通过添加还原剂和生物素，含有重组蛋白的CPFB标签可以与M6分离。这一特点使得M6系统适用于可重复使用芯片、生物反应器和其他应用的开发。本研究的第一个目标是测试我们是否可以使用M6系统开发可重复使用的芯片。该系统的一个缺点是CPFB标签必须被生物素化。如果可以省略生物素化步骤，则该系统可以更加通用。因此，第二个研究目标是用另一种不需要生物素化就能与M6结合的标签代替CPFB标签。将半胱氨酸残基引入该标签，并检查标签与M6之间二硫键的形成。第三个目的是看是否有可能通过蛋白质工程使单体链霉亲和素具有与天然链霉亲和素一样好的生物素结合能力。第二个项目的长期目标是使用一种非致病细菌枯草芽孢杆菌作为表面展示系统，在细胞表面展示生物分子。所展示的分子可以是消化存在于我们环境中的聚合物的酶，也可以是用于疫苗制备的抗原。我们将研究枯草芽孢杆菌中细胞壁蛋白与细胞壁及其底物共价连接的酶（分选酶）。这些壁结合蛋白的生理作用将被确定。基于本研究产生的知识，将开发一个表面显示系统。

This proposal has two projects. The first project focuses on streptavidin engineering. Streptavidin is a four-subunit, biotin binding protein. Because of the tight binding, streptavidin serves as an immobilization agent for biotinylated proteins. A new streptavidin designated M6 has been designed by us. It has only one subunit per molecule and can bind biotinylated proteins in a reversible manner. M6 has a single cysteine residue and can form a disulfide bond with recombinant proteins carrying a cysteine containing biotinylation peptide tag designated CPFB. Therefore, M6 has the ability to immobilize CPFB tag containing recombinant proteins. By the addition of a reducing agent and biotin, CPFB tag containing recombinant proteins can dissociate from M6. This feature makes the M6 system suitable for the development of reusable chips, bioreactors and other applications. The first objective for this study is to test whether we can develop reusable chips using the M6 system. One drawback for this system is that the CPFB tag has to be biotinylated. If the biotinylation step can be omitted, the system can be even more versatile. Therefore, the second research objective is to replace the CPFB tag with another tag that can bind to M6 without the requirement of biotinylation. A cysteine residue will be introduced to that tag and the formation of disulfide bond between the tag and M6 will be examined. The third objective is to see whether it is possible to make the monomeric streptavidin to have the biotin binding capability as good as the natural streptavidin through protein engineering. The long-term objective for the second project is to use a non-disease causing bacterium, Bacillus subtilis, as a surface display system to display biomolecules on the cell surface. The displayed molecules can be enzymes to digest polymers present in our environment or antigens for vaccine preparation. We will study enzymes (sortases) that can covalently link cell wall proteins to cell wall and their substrates in B. subtilis. The physiological roles of these wall bound proteins will be determined. A surface display system will be developed based on the knowledge generated from this study.