P-25 Expression and characterization of Ecm14, a putative metallocarboxypeptidase from Saccharomyces cerevisiae

Buller Hallway

Metallocarboxypeptidases are found in most organisms and function in the digestion and maturation of proteins. Ecm14 is a putative metallocarboxypeptidase found in the yeast Saccharomyces cerevisiae vacuole. There are a number of amino acids in the putative active site of Ecm14 that suggest a mechanism different from the typical carboxypeptidase. In order to investigate the enzymatic mechanism of Ecm14, expression of histidine-tagged Ecm14 protein was attempted in human HEK293T cell culture, S. cerevisiae, and baculovirus expression systems. No expression was detected in HEK293T cells in preliminary experiments. Expression in the yeast system resulted in insolubility of Ecm14, regardless of induction time, temperature, or inducer concentration. In contrast, following expression of Ecm14 in Sf9 cells using the baculovirus system, approximately 31% of Ecm14 was soluble and detected as a 40 kDa histidine-tagged protein by western blotting. Sf9-expressed Ecm14 was purified using metal affinity chromatography. No enzymatic activity could be detected for purified Ecm14 in the presence of substrate consisting of chromogenic 3-(2-furyl) acryloyl conjugated to a C-terminal dipeptide (Phe-Ala). Activation of Ecm14 by enzymatic removal of the prodomain is currently being pursued. Alternatively, Ecm14 may catalyze the cleavage of an unknown substrate or be an example of a catalytically-inactive protease-like protein.