Developing an Expression System for Recombinant Human Serine Protease 23, a Potential Target for Anti-cancer Treatment
2Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, USA
Correlative gene expression studies and genetic-based comparative models have identified PRSS23 gene, encoding serine protease 23, as a hallmark of several pathological conditions, including cancer. However, no information related to the biochemical or structural characterization of PRSS23 protein is currently available in the literature. To address this issue, we have engaged in creating a robust expression system for recombinant PRSS23. Based on the protein sequence, we hypothesized that PRSS23 is endogenously secreted and subsequently activated by furin cleavage, releasing the catalytically active C-terminal domain and an inactive N-terminal pro-domain. We started by generating various expression plasmids, encoding full-length PRSS23 or its catalytic domain, N-terminally fused to Small Ubiquitin-like Modifier (SUMO) for improved folding and solubility. These plasmids were transformed into the corresponding host organisms and the resulting colonies were screened by PCR. We then proceeded to establish and optimize the conditions for PRSS23 protein expression. High amounts of protein were obtained in the E. coli cytoplasm, in the form of insoluble inclusion bodies. Therefore, our current efforts are directed towards identifying optimal conditions for in vitro refolding. In P. pastoris expression system we have managed to detect a fragment of approximately 25 kDa, by Western blot analysis, indicating the production and activation of PRSS23 by furin cleavage. However, additional experiments are needed, using a more specific antibody, to confirm these results. Once the protein has been successfully produced, we will proceed to identify endogenous substrates, characterize its enzymatic activity and develop new therapeutic strategies targeting PRSS23 protein for anti-cancer treatment.