EXPRESSION IN ESCHERICHIA-COLI OF THE EXTRACELLULAR BASIC PROTEASE FROM DICHELOBACTER-NODOSUS

Dichelobacter nodosus, a Gram-negative obligate anaerobe and the causa tive agent of ovine footrot, secretes a number of extracellular protea ses, one of which is highly basic in nature. The gene (bprV) encoding this basic protease, from virulent strain 198, has been cloned and seq uenced. Clone pBR3KB contained the complete bprV gene which constituti vely expressed an active protease using its own promoter when cloned i n Escherichia coil. However, levels of protease expression were low an d unstable when the clone was expressed in liquid culture. A range of L coli strains were examined for stable expression; strains NH274 and SURE(TM) were found to be better hosts for stable expression than othe r commonly used E. coli host strains. Stabilization and enhancement of expression was achieved by deletion of the native promoter region and expression from plasmid promoter or promoters, and by modification of culture conditions. The recombinant protease obtained from E. coli wa s indistinguishable from the native enzyme in size, activity, isoelect ric point and immunological properties.