title: Evaluating secretion and surface attachment of SapA, an S-layer-associated metalloprotease of Caulobacter crescentus.
abstract: Caulobacter                         crescentus is used to display foreign peptides at high density as insertions into the surface (S)-layer protein (RsaA). Many recombinant RsaA proteins, however, are cleaved by SapA, a 71-kDa metalloprotease, suggesting a role in maintaining S-layer integrity. When overexpressed on a multicopy plasmid SapA was detected on the surface by fluorescent antibody only if RsaA and the O-side chain of LPS that mediates S-layer attachment were removed by mutation, indicating an outer membrane location beneath the S-layer. Secretion was mediated by the RsaA type 1 transporter since secretion was eliminated in transporter deficient strains or by C-terminal deletions in SapA (the presumed location of type 1 secretion signals). Secretion was required to become an active protease; mass spectrometry suggested this might be due to N-terminal processing during secretion, a feature shared with other type 1-secreted proteases. Overexpression leads to additional processing C-terminal to the protease domain, producing a 45-kDa protein. This was demonstrated to be self-processing. Deletion analysis revealed the C-terminal 100 amino acids were sufficient for anchoring and secretion. When protein G was fused to the last 238 amino acids of SapA it was secreted, surface attached and bound immunoglobulin, indicating potential for foreign protein display.
authors.affiliation: Department of Microbiology and Immunology, University of British Columbia, #2509-2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.
authors.forename: Lyngrace
authors.initials: L
authors.lastname: Gandham
authors.forename: John F
authors.initials: JF
authors.lastname: Nomellini
authors.forename: John
authors.initials: J
authors.lastname: Smit
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058947:Bacterial Secretion Systems; D016935:Caulobacter crescentus / Q000201:enzymology* / Q000235:genetics; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000378:metabolism*; D045726:Metalloproteases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C055764:S-layer proteins; D045726:Metalloproteases