Effects of MAT alpha deletions on protein secretion in Pichia pastoris

Pichia pastoris is a yeast known to efficiently express and secrete heterologous proteins. In this yeast, MAT alpha is a signal that can direct protein secretion. Its effect on secretion can be tested using reporter genes, whose protein products can readily be measured. The goal of our project was to create specific deletions in the MAT alpha secretion signal and to determine their effects on secretion. Mutant constructs were made via site-directed mutagenesis, and the reporter genes tested were horseradish peroxidase (HRP) and lipase. We hypothesized that distinct deletions in the MAT alpha secretion signal should affect protein secretion differently, and these effects were tested by comparing secretion of the HRP and lipase proteins

Effects of MAT alpha deletions on protein secretion in Pichia pastoris

Pichia pastoris is a yeast known to efficiently express and secrete heterologous proteins. In this yeast, MAT alpha is a signal that can direct protein secretion. Its effect on secretion can be tested using reporter genes, whose protein products can readily be measured. The goal of our project was to create specific deletions in the MAT alpha secretion signal and to determine their effects on secretion. Mutant constructs were made via site-directed mutagenesis, and the reporter genes tested were horseradish peroxidase (HRP) and lipase. We hypothesized that distinct deletions in the MAT alpha secretion signal should affect protein secretion differently, and these effects were tested by comparing secretion of the HRP and lipase proteins

The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57–70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future