Improving Secretion Efficiency of Pichiaa pastoris by Mutagenesis of the Mat-Alpha Secretion Leader

Introduction: The yeast Pichia pastoris is extremely useful to the biotechnology industry because it can efficiently express and secrete foreign proteins. This ability allows manufacturers to produce and purify medically important proteins such as angiostatin and endostatin. Previous research has show that a small leader peptide, known as Mat alpha mating factor (Matα), can be attached to the N- terminus of a protein construct, where it acts as an “address label” that signals the Pichia cell to secrete the foreign protein into the surrounding culture medium.Objective: The goal of this project was to develop a stronger version of Matα, which would result in higher yields and more efficient secretion of heterologous proteins from Pichia.Methods: Using computer modeling of the Matα secondary structure, I discovered several sequences of amino acids in this gene which might play a role in the secretion pathway. I then used site-directed mutagenesis to create mutant Pichia strains which lacked these target sequences and tested their secretion efficiency using a reporter-gene assay.Results and Conclusions: Several mutant strains were found to have significantly greater secretion levels than wild-type Pichia pastoris cultures, indicating that the targeted amino acid sequences could play an essential role in the export pathway