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

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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.