The methylotrophic yeast, Pichia pastoris, is the most successful and favored microbial eukaryotic expression system for the production of recombinant proteins for biopharmaceutical or industrial purposes. P. pastoris has the ability to produce foreign proteins at high levels extracellularly, and since it secretes few endogenous proteins, this ability eliminates the need for expensive purification costs. It also combines the ease of genetic manipulation with rapid growth to high cell densities and provides complex posttranslational modifications. The most commonly utilized secretion signal leader in P. pastoris is the MATα prepro signal leader, originally found in S. cerevisiae. However, because some proteins cannot be secreted efficiently by P. pastoris, strategies to enhance secretion efficiency have involved the modification of the MATα prepro secretion signal leader. The study focuses on using site-directed mutagenesis of specific sets of amino acids of MATα prepro secretion leader to evaluate the correlation between secondary structure and secretion level. MATα pro-HRP mutants were created, in order to analyze the export of heterologous proteins in P. pastoris. In addition, structural analysis through circular dichroism was performed on mutant MATα pro-peptides to evaluate differences in secondary structure as a result of the mutagenesis. Mutants, pSC6 (Δ57-65) and pSC7 (Δ66-70) did not generate the same HRP secretion level as Δ57-70. In addition, a new proposed model of MATα pro-peptide signal leader was created. This new model suggests that the N and C terminus of MATα pro-peptide need to be presented correctly for proper interaction with secretion machinery and for efficient protein secretion. With these analyses, optimization of secretion systems can be achieved to impact the fields of science, industry, healthcare, and economics worldwide.

Pichia pastoris is a methylotrophic yeast that has been used in both research and industrial settings for recombinant protein expression due to the ease of genetically modifying its genome, its ability to grow to large densities in inexpensive media, and its capability to perform posttranslational modifications. Multiple tools such as the cis -acting factors MATα secretion signal and MBP fusion partners, and trans-acting modifications such as the bgs mutants have increased heterologous protein secretion. Although these techniques have already been used, their effects on the protein secretory pathway have yet to be elucidated. In this study, fluorescence microscopy was used to compare the localization of proteins expressed with the mutated MATα with the deletion of amino acids 57-70 to the wild type MATα secretion signal. Additional fluorescence microscopy was completed to visualize the localization of MBP-EGFP and EGFP-MBP fusion proteins and their spatial relativity to organelle markers. EGFP-MBP was used to further distinguish the properties of multiple bgs mutants. Additionally, secreted lipase activity levels were evaluated in bgs13 strains expressing either the wild type or the mutated MAT&agr; signal peptide. The results indicated that regardless of their differences, the MATα secretion signals and bgs mutants transported their cargo proteins through similar pathways within the cells. The results of the MBP fusion proteins suggest that the arrangement of MBP significantly influences protein secretion and localization. Lastly, the bgs13 mutant with MATα secretion signals demonstrated that lipase activity increased additively when cis- and trans -acting mutations were combined. Ultimately, these results can provide better understanding of each modified factor and the protein sorting pathway, leading to potential techniques that optimize protein secretion in P. pastoris .