The structure and functions of proteins are highly reliant on their folding. Molecular chaperones assist with this process, and these chaperones may occur within the protein itself. Carboxypeptidase A1 (CPA1) is an enzyme that contains a prodomain that has been previously inferred to be an intra-molecular chaperone; however, the discovery of a related enzyme lacking a prodomain, carboxypeptidase O (CPO), questions the necessity of the prodomain for folding. The amino acid sequence of CPO was analyzed to find residues that were conserved and unique to CPO when compared with CPA1 and related carboxypeptidases. Four such residues were identified and found through site-directed mutagenesis and western blotting to be necessary for expression of CPO. In preparation for transferring these CPO residues to CPA1 by site-directed mutagenesis to examine their effect on CPA1 expression, PCR was used to insert an HA tag at the C-terminus of CPA1. Transfection of this plasmid encoding HA-tagged CPA1 into HEK293T cells resulted in expression of the protein, as determined by western blotting, and detection of enzymatic activity. Subsequent deletion of the prodomain, transferring of unique CPO residues to CPA1, and analysis of expression is ongoing. These studies will help us to determine the mechanism by which CPO folds in the absence of a prodomain, and so further our understanding of protein stability and folding.
Support or Funding Information
This work was supported by funds from Andrews University to MD and PJL.
