S12: Development of a protease activity tool kit for the identification and characterization of native Pichia pastoris proteases

A protease tool kit was assembled to detect protease activity, identify the responsible proteases, and provide direction in resolving the problem of product specific protease degradation in the yeast Pichia pastoris.  The tool kit was evaluated using two model proteins; the first a 19kDa therapeutic protein, the second a monoclonal antibody.  In the case of the therapeutic protein, use of a fluorescent casein substrate showed significant extracellular protease activity and increasing intracellular protease activity as the cultivation progressed.  Using a protease inhibitor screen, Chymostatin and Pepstatin A were found to decrease the product cleavage by 2 fold and 5 fold respectively.  Expression profiling identified both prb1 and pep4 transcripts as abundant in the cell culture, which correlated with the protease inhibitor screening results.  By knocking out the prb1 and pep4 genes as well as including protease inhibitors in the cultivation process, the product titer increased approximately 150 fold.  In the case of the monoclonal antibody, the degree of protease activity was again profiled using a fluorescent casein substrate.  This time, acrylamide zymography using the antibody as a substrate followed by mass spec based protein identification, was employed to characterize the proteases active in the cell culture.  A glycine carboxypeptidase, homologous to the S. cerevisiae carboxypeptidase S, was shown to cleave the antibody likely at the point of connection between the light chain and heavy chain.