DEVELOPMENT OF PROTEASE-DEFICIENT, GRAM-POSITIVE HOST

OUR LONG-TERM AIM IS TO DEVELOP A GRAM-POSITIVE ORGANISM FORTHE 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 SITE-DIRECTED DELETION OF ANY PROTEASE GENES IDENTIFIED IN THE PRESENT STUDY. MUTAGENESIS OF DOUBLE-PROTEASE-NEGATIVE STRAINS OF B. SUBTILIS, AND SCREENS OF OTHER BACILLUS SPECIES FOR PROTEASE-DEFICIENT STRAINS, WILL BE PERFORMED IN AN EFFORT TO DEVELOP A GRAM-POSITIVE HOST THAT DOES NOT DEGRADE SECRETORY PRODUCTS.