Expression in Escherichia coli of the extracellular basic protease from Dichelobacter nodosus.
Dichelobacter nodosus, a Gram-negative obligate anaerobe and the causative agent of ovine footrot, secretes a number of extracellular proteases, one of which is highly basic in nature. The gene (bprV) encoding this basic protease, from virulent strain 198, has been cloned and sequenced. Clone pBR3KB contained the complete bprV gene which constitutively expressed an active protease using its own promoter, when cloned in Escherichia coli. However, levels of protease expression were low and unstable when the clone was expressed in liquid culture. A range of E. coli strains were examined for stable expression; strains NH274 and SURE were found to be better hosts for stable expression than other 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 was indistinguishable from the native enzyme in size, activity, isoelectric point and immunological properties.
Vaughan
P R
PR
CSIRO Division of Biomolecular Engineering, Parkville, Victoria, Australia.
Wang
L F
LF
Stewart
D J
DJ
Lilley
G G
GG
Kortt
A A
AA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001439:Bacteroides / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000145:classification / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015967:Gene Expression Regulation, Viral; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013604:T-Phages / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; C078345:extracellular basic protease, Dichelobacter nodosus