High-temperature cultivation of recombinant Pichia pastorisincreases endoplasmic reticulum stress and decreases production of human interleukin-10

Microbial Cell Factories

Research

Yugang Guo¹  Lu Yang¹  Ming Jiang¹  Wenyao Kang¹  Yongjun Zhong¹  Jiajia Ma¹  Fang Fang¹  Dong Wang¹  Rui Li¹  Weihua Xiao¹  Jie Sun¹ 

[1] The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, China;Hefei National Laboratory for Physical Sciences at Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China;

关键词: High-temperature cultivation;    Misfolded protein;    ER stress;    Unfolded protein response;    ER-phagy;    Pichia pastoris;   

DOI  :  10.1186/s12934-014-0163-7

 received in 2014-08-09, accepted in 2014-11-13,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundThe yeast Pichia pastoris (P. pastoris) has become a popular `cell factory' for producing heterologous proteins, but production widely varies among proteins. Cultivation temperature is frequently reported to significantly affect protein production; however, the underlying mechanisms of this effect remain unclear.ResultsA P. pastoris strain expressing recombinant human interleukin-10 (rhIL-10) under the control of the AOX1 promoter was used as the model in this study. This system shows high-yield rhIL-10 production with prolonged methanol-induction times when cultured at 20°C but low-yield rhIL-10 production and higher cell death rates when cultured at 30°C. Further investigation showed that G3-pro-rhIL10, an immature form of rhIL-10 that contains the glycosylation-modified signal peptide, remained in the ER for a prolonged period at 30°C. The retention resulted in higher ER stress levels that were accompanied by increased ROS production, Ca2+ leakage, ER-containing autophagosomes, shortened cortical ER length and compromised induction of the unfolded protein response (UPR). In contrast, G3-pro-rhIL10 was quickly processed and eliminated from the ER at 20°C, resulting in a lower level of ER stress and improved rhIL-10 production.ConclusionsHigh-temperature cultivation of an rhIL-10 expression strain leads to prolonged retention of immature G3-pro-rhIL10 in ER, causing higher ER stress levels and thus greater yeast cell death rates and lower production of rhIL-10.

【 授权许可】

Unknown   
© Zhong et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.