SBIR-STTR Award

Our long-term aim is to develop a gram-positive organism for the expression and secretion of medically and industrially useful proteins. One major obstacle to the successful development of such a system is the proteolytic degradation of secreted products. Site-directed deletion mutations constructed in vitro have eliminated the two major extracellular proteases of B. subtilis. Although significantly diminished, proteolytic degradation of secreted products by residual protease remains problematic.Characterization of residual protease activity will be performed preliminary to cloning and sitedirected deletion of any protease genes identified in the present study. Mutagenesis of doubleprotease-negative strains of B. subtilis, and screens of other Bacillus species for proteasedeficient strains, will be performed in an effort to develop a gram-positive host that does not degrade secretary products.National Institute of Allergy and Infectious Diseases (NIAID)

Applied Microbiology, Inc. (AMBI) has been developing Bacillus subtilis and related species as a host system for the production of recombinant protein products. B. subtilis has several advantages over E. coli (e.g., it secretes protein products, is nonpathogenic, and does not produce endotoxins). AMBI has previously inactivated the alkaline and neutral protease (AP and NP) genes of B. subtilis. Work done under the Phase I proposal identified and characterized the residual proteases, that together account for all remaining extracellular protease activity in the AP/NP strains constructed at AMBI. Under the Phase II proposal, AMBI will clone and inactivate the residual protease genes to produce a host free of extracellular protease activity. A means of expressing recombinant products throughout growth and in stationary cultures by the use of appropriate promoters will also be developed. This will greatly increase product yield in protease free hosts. In addition, a method to produce accurately processed recombinant proteins in B. subtilis will be determined.National Institute of Allergy and Infectious Diseases (NIAID)