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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0163-7) contains supplementary material, which is available to authorized users.

Co-expressing GroEL–GroES, Ssa1–Sis1 and Bip–PDI chaperones for enhanced intracellular production and partial-wall breaking improved stability of porcine growth hormone

“…To further increase the yield of pGH, the optimal temperature, pH and culture time for expression of pGH protein in Pichia pastoris were investigated by single factor experiments. Previous findings showed that low temperature induction resulted in more conducive to the expression of heterologous proteins in Pichia pastoris [52,53]. In our study, the expression of pGH protein in Pichia pastoris was highest at 20 °C, pH 6.0, for 48 h, which mirrored the finding that mRNA is more stable at the low temperature [54].…”

Optimized expression of the Starmerella bombicola lactone esterase in Pichia pastoris through temperature adaptation, codon-optimization and co-expression with HAC1

“…By reducing the induction temperature, we decrease the rate of protein synthesis, and give time for newly translated recombinant protein to fold properly. According to other literature examples, lowering induction temperature does not only lead to a higher expression, it can also lead to a more active protein [22] and an increase in cell viability [23,24]. Indeed, transcriptomic [25] and proteomic [26] analyses of Pichia pastoris recombinant protein expression at a lower temperature showed a strong decrease in folding stress, noticed by the decrease of chaperones and other folding-related proteins.…”

“…More precisely, the small variation in cell mass could therefore not be responsible for the observed increases in yield. Indeed, Zhong et al also observed that, during methanol induction, there was no statistical influence of a lower temperature on the growth curve of recombinant Pichia pastoris [23]. Although their experiment used an induction temperature of 20°C, it is remarkable that these results hold true for 16°C as well.…”

WHICH WAY DO YOU PREFER TO DEATH ? Cell Death and Beyond

“…Er-phagy has two subtypes, these subtypes are macro-ER-phagy and micro-ER-phagy which does not use autophagic organelles and machinery [123]. ER-phagy which selectively seizes protein aggregates containing ER fragments and returns the ER to its normal size once folding stress subsides, is suggested to provide a survival advantage in response to ER stress [124]. ER-phagy occurs after extended times of starvation or ER stress [125].…”