Enhanced Production of Recombinant Secretory Proteins in Pichia pastoris by Optimizing Kex2 P1’ site

Song Yang,Donghai Wu,Pentao Liu,Yulai Zhou,Yu Wang,Ye Kuang,Yuehong Liu,Zhiwu Jiang,Xiaoyan Hui,Qingyang Huang,Peng Li,Hongbo Li,Shiwu Li,Aimin Xu

doi:10.1371/journal.pone.0075347

Abstract

Pichia pastoris is one of the most widely used expression systems for the production of recombinant secretory proteins. Its universal application is, however, somewhat hampered by its unpredictable yields for different heterologous proteins, which is now believed to be caused in part by their varied efficiencies to traffic through the host secretion machinery. The yeast endoprotease Kex2 removes the signal peptides from pre-proteins and releases the mature form of secreted proteins, thus, plays a pivotal role in the yeast secretory pathways. In this study, we found that the yields of many recombinant proteins were greatly influenced by Kex2 P1' site residues and the optimized P1’s amino acid residue could largely determine the final amount of secretory proteins synthesized and secreted. A further improvement of secretory yield was achieved by genomic integration of additional Kex2 copies, which again highlighted the importance of Kex2 cleavage to the production of recombinant secretory proteins in Pichia yeast.

Highlights

Protein based biopharmaceuticals make up the largest and fastest growing part of global top selling drugs [1,2]
Pichia pastoris is one of the most commonly used expression hosts for production of heterologous secretory proteins [3], thanks mainly to a highly efficient and tightly regulated expression system based on the promoter of the alcohol oxidase 1 gene (AOX1), high levels of protein products being secreted into almost protein-free media as well as its capacity of carrying out correct folding and post-translational modification for mammalian proteins [4,5,6,7,8]
The P. pastoris recombinants were first grown in BMGY (1% yeast extract, 2% peptone, 100 mM potassium phosphate, 1.34% yeast nitrogen base (YNB), 4×10-5% biotin, 1% glycerol) medium to reach higher biomass and induced in BMMY medium which contains the same ingredients as BMGY except the replacement of glycerol with methanol, as detailed in EasySelectTM Pichia expression kit user manual

Summary

Introduction

Protein based biopharmaceuticals make up the largest and fastest growing part of global top selling drugs [1,2]. Pichia pastoris is one of the most commonly used expression hosts for production of heterologous secretory proteins [3], thanks mainly to a highly efficient and tightly regulated expression system based on the promoter of the alcohol oxidase 1 gene (AOX1), high levels of protein products being secreted into almost protein-free media as well as its capacity of carrying out correct folding and post-translational modification for mammalian proteins [4,5,6,7,8]. The strategies to improve the efficiency of this secretion machinery and the specific components within this complex system that may serve as a viable target for engineering remain elusive

Methods

Results

Conclusion