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Characterization of the secretion and anchoring domains of Caulobacter crescentus SapA metalloprotease

University of British Columbia

The Caulobacter crescentus type I secretion system can be used to display foreign peptides at high density on the bacterium’s surface as part of the S-layer. Certain recombinant proteins, however, are subject to proteolytic cleavage by SapA, a unique S-layer associated metalloprotease. SapA (71 kDa) is an unstable protein that breaks down to a 45-kDa product when over-expressed. It needs to be secreted before it can become an active enzyme that anchors to the cell. A point mutation adjacent to the protease’s active site reduced SapA processing, indicating that SapA is self-processing. The last 10 and 50 amino acids were removed and prevented secretion, indicating SapA was a type I secreted protein. Further, SapA secretion was blocked in an S-layer type I secretion deficient strain. Lack of secretion prevents this protease from becoming an active enzyme evidenced by the type I defective clones, which are not processed at all. The last 100 amino acids of the protease are sufficient for anchoring, as determined by immunofluorescence. Interestingly, SapA could be detected on the cell surface by immunofluorescence only in an S-layer negative, O-antigen deficient strain. This suggests that SapA is localized on the cell membrane, beneath the S-layer and is hidden by smooth LPS. A fusion protein, containing a 242 amino acid protein G peptide attached to the last 238 amino acids of SapA secreted and anchored to the cell surface of C. crescentus. This fusion was detectable an anti-IgG antibody. SapA is the first identified self-processing protease that uses its C-terminus for both type I secretion and anchoring.

Text

2010-05-18

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/24820

Microbiology and Immunology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/