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.
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Acknowledgments
We thank Sadeem Fayed for production of pET21b(+)-sap and aspects of anti-SapA antibody production and Janny Lau for construction of JS2007. This work was supported by a grant from the Natural Sciences Engineering and Research Council of Canada to J.S.
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Communicated by Eriko Takano.
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Gandham, L., Nomellini, J.F. & Smit, J. Evaluating secretion and surface attachment of SapA, an S-layer-associated metalloprotease of Caulobacter crescentus . Arch Microbiol 194, 865–877 (2012). https://doi.org/10.1007/s00203-012-0819-9
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DOI: https://doi.org/10.1007/s00203-012-0819-9