title: High-temperature cultivation of recombinant Pichia pastoris increases endoplasmic reticulum stress and decreases production of human interleukin-10.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  A 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, Ca(2+) 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.
  
  
  CONCLUSIONS
  High-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.
authors.affiliation: 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. yjzhong@mail.ustc.edu.cn.|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. yjzhong@mail.ustc.edu.cn.
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Zhong
authors.affiliation: 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. Yanglulu@mail.ustc.edu.cn.|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. Yanglulu@mail.ustc.edu.cn.
authors.forename: Lu
authors.initials: L
authors.lastname: Yang
authors.affiliation: 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. gmp@ustc.edu.cn.|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. gmp@ustc.edu.cn.
authors.forename: Yugang
authors.initials: Y
authors.lastname: Guo
authors.affiliation: 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. fangfang@mail.ustc.edu.cn.|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. fangfang@mail.ustc.edu.cn.
authors.forename: Fang
authors.initials: F
authors.lastname: Fang
authors.affiliation: 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. wdwade@mail.ustc.edu.cn.|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. wdwade@mail.ustc.edu.cn.
authors.forename: Dong
authors.initials: D
authors.lastname: Wang
authors.affiliation: 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. lirui@mail.ustc.edu.cn.|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. lirui@mail.ustc.edu.cn.
authors.forename: Rui
authors.initials: R
authors.lastname: Li
authors.affiliation: 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. jiangm86@mail.ustc.edu.cn.|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. jiangm86@mail.ustc.edu.cn.
authors.forename: Ming
authors.initials: M
authors.lastname: Jiang
authors.affiliation: 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. kangwy@mail.ustc.edu.cn.|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. kangwy@mail.ustc.edu.cn.
authors.forename: Wenyao
authors.initials: W
authors.lastname: Kang
authors.affiliation: 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. jjma@ustc.edu.cn.|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. jjma@ustc.edu.cn.
authors.forename: Jiajia
authors.initials: J
authors.lastname: Ma
authors.affiliation: 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. sj519@ustc.edu.cn.|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. sj519@ustc.edu.cn.
authors.forename: Jie
authors.initials: J
authors.lastname: Sun
authors.affiliation: 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. xiaow@ustc.edu.cn.|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. xiaow@ustc.edu.cn.
authors.forename: Weihua
authors.initials: W
authors.lastname: Xiao
mesh_terms: D059865:Endoplasmic Reticulum Stress; D006358:Hot Temperature; D006801:Humans; D016753:Interleukin-10 / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D056811:Unfolded Protein Response
chemical_list: C508609:IL10 protein, human; D011994:Recombinant Proteins; D016753:Interleukin-10