当前位置： SCI文献检索 > Journal of Proteomics期刊下所有文献 > Quantitative iTRAQ LC-MS/MS proteomics reveals the cellular response to heterologous protein overexpression and the regulation of HAC1 in Pichia pastoris.

Quantitative iTRAQ LC-MS/MS proteomics reveals the cellular response to heterologous protein overexpression and the regulation of HAC1 in Pichia pastoris.

Abstract：

UNLABELLED:The methylotrophic yeast Pichia pastoris is an attractive platform for a plethora of recombinant proteins. There is growing evidence that host cells producing recombinant proteins are exposed to a variety of cellular stresses resulting in the induction of the unfolded protein response (UPR) pathway. At present, there is only limited information about the cellular reactions of the host cells at the level of the proteome, especially with regard to recombinant protein secretion. Here we monitored xylanase A secretion from Bacillus halodurans C-125 (xynA) in P. pastoris, using strains containing different copy numbers of the gene encoding xylanase A and co-overexpressing the gene encoding the UPR-regulating transcription factor HAC1 by applying a quantitative proteomics approach (iTRAQ-LC-MS/MS). Many important cellular processes, including carbon metabolism, stress response and protein folding are affected in the investigated conditions. Notably, the analysis revealed that strong over-expression of xynA can efficiently improve protein production but simultaneously cause an unfolded protein burden with a subsequent induction of the UPR. This limits the further improvement of protein production levels. Remarkably, constitutive expression of the gene encoding HAC1 lessens the unfolded protein burden by attenuating protein synthesis and increasing ER protein folding efficiency which is beneficial for protein secretion. BIOLOGICAL SIGNIFICANCE:Pichia pastoris expression systems have been successfully used for over 20years in basic research and in the biotechnology industry for the production and secretion of a wide range of recombinant proteins. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. It has become obvious that many protein products can lead to severe stress on the host cell when being over-expressed, thus limiting the potential yield. Detailed understanding of the physiological responses to such stresses gives rise to engineering of host cells that can better cope with the stress factors. Therefore, the regulatory mechanism of heterologous protein secretion by quantitative mass-spectrometry (MS) proteomics is a growing field and an important endeavor in improving protein annotation. Many important cellular processes, including carbon and amino acid metabolism, stress response and protein folding are affected in the over-expression strains. This data represent a first step towards a systems wide approach to assess the response with recombinant protein induced stress in P. pastoris.

journal_name

J Proteomics

journal_title

Journal of proteomics

authors

Lin XQ,Liang SL,Han SY,Zheng SP,Ye YR,Lin Y

doi

10.1016/j.jprot.2013.06.031

subject

Has Abstract

pub_date

2013-10-08 00:00:00

pages

58-72

eissn

1874-3919

issn

1876-7737

pii

S1874-3919(13)00379-5

journal_volume

pub_type

杂志文章

在线工具

Quantitative iTRAQ LC-MS/MS proteomics reveals the cellular response to heterologous protein overexpression and the regulation of HAC1 in Pichia pastoris.