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P-26 Purification and characterization of Ecm14, a fungal pseudopeptidase
Celebration of Research and Creative Scholarship
  • Peter J Lyons, Andrews University
  • Matthew J Schott, Andrews University
Presenter Status
Department of Biology
Second Presenter Status
Graduate Student, Department of Biology
Preferred Session
Poster Session
Start Date
30-10-2015 2:00 PM
End Date
30-10-2015 3:00 PM
Presentation Abstract

Peptidases are enzymes important for hydrolysis of peptide bonds and hence degradation or modification of proteins. Many families of peptidases also contain inactive enzyme homologs that are predicted to be inactive due to the alteration of critical active site amino acids. The M14 family of metallocarboxypeptidases is a large family of peptidases made up of 23 members in humans, although three members are predicted to be inactive. The important eukaryotic model system, the yeast Saccharomyces cerevisiae, has only one metallocarboxypeptidase homolog, Ecm14, and this sole homolog is predicted to be inactive. An analysis of available fungal amino acid sequences showed that the Ecm14 gene was present and conserved in ascomycete fungi only. In order to characterize the function of Ecm14, we expressed a C-terminally histidine tagged version of Ecm14 using the Sf9/baculovirus system. The protein was secreted and subsequently purified by metal affinity chromatography. Incubation of Ecm14 with trypsin or chymotrypsin resulted in the specific removal of the Ecm14 prodomain. The mature form of Ecm14 lacked any detectable enzymatic activity, but was able to prevent the cleavage of standard substrates by an active metallocarboxypeptidase. We propose that Ecm14 may perform a regulatory function through binding to specific C-terminal amino acids, thus blocking their cleavage by active carboxypeptidases.

Acknowledgments

We acknowledge funding for this work from an Andrews University Faculty Research Grant.

Citation Information
Peter J Lyons and Matthew J Schott. "P-26 Purification and characterization of Ecm14, a fungal pseudopeptidase" (2015)
Available at: http://works.bepress.com/peter_lyons/9/