ABSTRACT
The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation, the ability to provide complex posttranslational modifications and the capacity for efficient protein secretion. The most successful and commonly used secretion signal leader in Pichia pastoris has been the alpha mating factor (MATα) prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently, leading to strategies to enhance secretion efficiency by modifying the secretion signal leader. Based on a Jpred secondary structure prediction and knob-socket modeling of tertiary structure, numerous deletions and duplications of the MATα prepro leader were engineered to evaluate the correlation between predicted secondary structure and the secretion level of the reporters horseradish peroxidase (HRP) and Candida antarctica lipase B. In addition, circular dichroism analyses were completed for the wild type and several mutant pro-peptides to evaluate actual differences in secondary structure. The results lead to a new model of MATα pro-peptide signal leader, which suggests that the N and C-termini of MATα pro-peptide need to be presented in a specific orientation for proper interaction with the cellular secretion machinery and for efficient protein secretion.
Subject(s)
Fungal Proteins/genetics , Mating Factor/genetics , Peptides/genetics , Pichia/genetics , Recombinant Fusion Proteins/genetics , Amino Acid Sequence , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Horseradish Peroxidase/genetics , Horseradish Peroxidase/metabolism , Lipase/genetics , Lipase/metabolism , Mating Factor/chemistry , Mating Factor/metabolism , Models, Molecular , Mutation , Peptides/chemistry , Peptides/metabolism , Pichia/metabolism , Protein Precursors/chemistry , Protein Precursors/genetics , Protein Precursors/metabolism , Protein Sorting Signals/genetics , Protein Structure, Secondary , Recombinant Fusion Proteins/metabolism , Sequence DeletionABSTRACT
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.
