Overexpression, purification, and refolding of a Porphyromonas gingivalis cysteine protease from Escherichia coli.
This paper describes the overexpression of the Rgp-1 (arginine) protease domain from Porphyromonas gingivalis. This protease and the related Kgp (lysine) protease, both of which display trypsin-like specificity, have been implicated as major virulence factors and may play a significant role in the etiology of periodontal disease. Both Rgp-1 and Kgp are initially translated as polyproteins, each containing a protease domain and multiple adhesin domains. The Rgp-1 protease domain was expressed in E. coli, purified, refolded, and assayed for activity. These expression studies demonstrated that prior to the formation of inclusion bodies in the E. coli cytoplasm, the protease was proteolytically active and could hydrolyze a specific synthetic substrate. When the Rgp-1 protease domain was purified from inclusion bodies and refolded, it was found to be autolytically active and displayed specific catalytic activity. This is the first report on the expression and purification of active Rgp-1 from E. coli. Polyclonal antisera raised against recombinant protein recognized the native form of the protease in the P. gingivalis strain W50, indicating that the recombinant protein contained some of the antigenic determinants of the native protease.
Margetts
M B
MB
Research and Development Division, CSL Ltd., 45 Poplar Road, Parkville, Victoria, 3052, Australia. Mai_Margetts@csl.com.au
Barr
I G
IG
Webb
E A
EA
D018829:Adhesins, Bacterial; D015153:Blotting, Western; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007106:Immune Sera; D002479:Inclusion Bodies / Q000737:chemistry*; D016966:Porphyromonas gingivalis / Q000737:chemistry*; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D018829:Adhesins, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D007106:Immune Sera; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases