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 and localized to the vacuole. There are a number of amino acids in the putative active site of ECM14 that suggest a catalytic mechanism different from the typical carboxypeptidase. In order to investigate the enzymatic mechanism of ECM14, the protein was histidine-tagged and over-expressed in S. cerevisiae. Western blotting of protein extracts resulted in a 40 kDa histidine-tagged protein, intermediate between the predicted sizes of the proenzyme (50 kDa) and the mature enzyme (35 kDa). Proteinase A, the endopeptidase predicted to be responsible for maturation of ECM14, was co-expressed with ECM14, but resulted in no change in ECM14 size, suggesting that the mature form may already be present. Overexpressed ECM14 protein was insoluble. To obtain soluble protein, ECM14 expression was induced at low temperature, for varying lengths of time, and at lower inducer concentrations. No change in solubility of ECM14 was observed under any of these conditions. Further experiments to obtain soluble protein will make use of an insect cell expression system and refolding from a denatured state.
Available at: http://works.bepress.com/peter_lyons/10/