Improving the secretion of a methyl parathion hydrolase in Pichia pastoris by modifying its N-terminal sequence.

Ping Wang,Hu Jiang,Ningfeng Wu,Jian Tian,Xiaoyu Chu,Lu Huang,Israel Silman

doi:10.1371/journal.pone.0096974

Abstract

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 (N66-MPH, D10-MPH, and N9-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 D10-MPH was increased to 0.21 U/mL, while that of N9-MPH was enhanced to 0.16 U/mL. Although N66-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.

Highlights

Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express heterologous proteins for use in research, as well as industrial and pharmaceutical applications [1]
In the P. pastoris expression system, signal peptides are commonly included in commercially available vectors, and so can be attached to the recombinant protein, causing it to be exported from the cell
Three-dimensional Models of methyl parathion hydrolase (MPH), OPHC2, and N9-MPH The 3D structures of MPH, OPHC2, and N9-MPH were modeled based on the crystal structure of Pseudomonas sp

Summary

Introduction

Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express heterologous proteins for use in research, as well as industrial and pharmaceutical applications [1]. The preferential secretion of recombinant proteins allows for the direct isolation of target proteins from culture media, eliminating the need for high-cost, low-yield cell disruption. This feature limits the toxicity issues resulting from intracellular accumulation of target proteins [2]. Because P. pastoris secretes few intrinsic proteins, the recombinant protein forms the major polypeptide species found in the extracellular growth medium, which facilitates purification of the heterologous protein [3]

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