Characterization of the Role of BGS13 in the Secretory Mechanism of Pichia pastoris

The methylotrophic yeast Pichia pastoris has been utilized for heterologous protein expression for over thirty years. Because P. pastoris secretes few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, some recombinant proteins intended for secretion are retained within the cell. A mutant strain isolated in our lab, containing a disruption of the BGS13 gene, displayed elevated levels of secretion for a variety of reporter proteins. The Bgs13 peptide is similar to the S. cerevisiae protein kinase C (Pkc1) protein but its specific mode(s) of action is currently unclear. To illuminate differences in the secretion mechanism between wt and the bgs13 strains, we determined that the disrupted bgs13 gene still expressed a truncated protein that had reduced protein kinase C activity and a different location in the cell compared to the wt protein. Because the Pkc1p of baker9s yeast played a significant role in cell wall integrity, we investigated the sensitivity of the mutant strain9s cell wall to growth antagonists and extraction by DTT, determining that the bgs13 cell wall suffered from inherent structural problems although its porosity was normal. A proteomic investigation of the bgs13 secretome and cell wall-extracted peptides demonstrated that, compared to its wt parent, the bgs13 strain also displayed increased release of an array of normally secreted, endogenous proteins as well as ER resident chaperone proteins, intimating that Bgs13p helps regulate the Unfolded Protein Response (UPR) and protein sorting on a global scale. IMPORTANCE The yeast Pichia pastoris is used as a host system for the expression of recombinant proteins. Many of these products are currently on the market or in late stage development, including antibodies, vaccine antigens and therapeutic proteins like insulin. However one major weakness is that sometimes these proteins are not secreted out of the yeast cell efficiently, which impedes and raises the cost of purification of these vital proteins. Our lab has isolated a mutant strain of Pichia pastoris that shows enhanced secretion of many proteins. The mutant produces a modified version of the Bgs13 protein. Our goal is to understand how the change in the Bgs13 protein function leads to improved secretion. Once the Bgs13 mechanism is illuminated, we should be able to apply this understanding to engineer new P. pastoris strains that efficiently produce and secrete life-saving recombinant proteins, providing medical and economic benefits.