Improving the Secretion of a Methyl Parathion Hydrolase in Pichia pastoris by Modifying Its N-Terminal Sequence

Pichia pastoris is commonly used to express and secrete target proteins, although not all recombinant proteins can be successfully produced. In this study, we used methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 as a model to study the importance of the N-terminus of the protein for its secretion. While MPH can be efficiently expressed intracellularly in P. pastoris, it is not secreted into the extracellular environment. Three MPH mutants (N(66)-MPH, D(10)-MPH, and N(9)-MPH) were constructed through modification of its N-terminus, and the secretion of each by P. pastoris was improved when compared to wild-type MPH. The level of secreted D(10)-MPH was increased to 0.21 U/mL, while that of N(9)-MPH was enhanced to 0.16 U/mL. Although N(66)-MPH was not enzymatically active, it was secreted efficiently, and was identified by SDS-PAGE. These results demonstrate that the secretion of heterologous proteins in P. pastoris may be improved by modifying their N-terminal structures.