An exhaustive body of data has been generated that indicates the importance of efforts to further develop cyanovirin-N (CV-N) as an anti-HIV-1 microbicide. It is a highly potent inhibitor of cell-free and cell-to-cell transmission of HIV-1 infection, with no observable toxic effect on host cells. The primary obstacle to advancing CV-N as a microbicide is the fact that it is currently difficult to produce in quantities required for proper preclinical study and clinical evaluation. As a recombinant protein, CV-N must be produced by means of an in vivo recombinant expression system. Our central hypothesis in this Project is that being of prokaryotic origin, CV-N should be highly compatible with bacteria-based expression systems, and its unique biochemical properties make it feasible to apply an array of purification technologies that typically would not be appropriate in efforts to obtain a bioactive recombinant protein. Therefore, we have defined a set of Specific Aims that are designed to: (1) comprehensively study the regulation of CV-N expression in bacterial systems; (2) exploit the unique biochemical properties of CV-N in the development of a high yield purification scheme; (3) determine the relevance of specific growth parameters to the expression of CV-N in larger scale fermentation; and (4) test if alternative forms of CV-N can be engineered that either enhance production or HIV-1 inhibitory activity. These efforts will be achieved through extensive interaction between the research team of this project, and the other Research Projects and Cores of the Program.
