Fate of the UPR marker protein Kar2/Bip and autophagic processes in fed-batch cultures of secretory insulin precursor producing Pichia pastoris
Creators: name: Roth, Gustavo
name: Vanz, Ana Leticia
name: Lünsdorf, Heinrich
name: Nimtz, Manfred
name: Rinas, Ursula
Publisher: name: London : BioMed Central Ltd.
Year: 2018
Subjects: subject: Autophagy
subject: Kar2/Bip
subject: Pichia pastoris
subject: Unfolded protein response
subject: fungal protein
subject: fungal protein Kar2
subject: glucose regulated protein 78
subject: glycerol
subject: insulin
subject: insulin precursor
subject: methanol
subject: unclassified drug
subject: Article
subject: autophagy
subject: cell structure
subject: controlled study
subject: fed batch culture
subject: independent variable
subject: insulin release
subject: insulin synthesis
subject: Komagataella pastoris
subject: nonhuman
subject: protein degradation
subject: unfolded protein response
subject: Dewey Decimal Classification::600 | Technik::610 | Medizin, Gesundheit
subject: Dewey Decimal Classification::500 | Naturwissenschaften::570 | Biowissenschaften, Biologie

description: Background: Secretory recombinant protein production with Pichia (syn. Komagataella) pastoris is commonly associated with the induction of an unfolded protein response (UPR) usually apparent through increased intracellular levels of endoplasmic reticulum (ER) resident chaperones such as Kar2/Bip. During methanol-induced secretory production of an insulin precursor (IP) under industrially relevant fed-batch conditions the initially high level of intracellular Kar2/Bip after batch growth on glycerol unexpectedly declined in the following methanol fed-batch phase misleadingly suggesting that IP production had a low impact on UPR activation. Results: Analysis of the protein production independent level of Kar2/Bip revealed that high Kar2/Bip levels were reached in the exponential growth phase of glycerol batch cultures followed by a strong decline of Kar2/Bip during entry into stationary phase. Ultra-structural cell morphology studies revealed autophagic processes (e.g. ER phagy) at the end of the glycerol batch phase most likely responsible for the degradation of ER resident chaperones such as Kar2/Bip. The pre-induction level of Kar2/Bip did not affect the IP secretion efficiency in the subsequent methanol-induced IP production phase. During growth on methanol intracellular Kar2/Bip levels declined in IP producing and non-producing host cells. However, extracellular accumulation of Kar2/Bip was observed in IP-producing cultures but not in non-producing controls. Most importantly, the majority of the extracellular Kar2/Bip accumulated in the culture supernatant of IP producing cells as truncated protein (approx. 35 kDa). Conclusions: Rapid growth leads to higher basal levels of the major UPR marker protein Kar2/Bip independent of recombinant protein production. Entry into stationary phase or slower growth on poorer substrate, e.g. methanol, leads to a lower basal Kar2/Bip level. Methanol-induced secretory IP production elicits a strong UPR activation which counteracts the reduced UPR during slow growth on methanol. The major ER chaperone Kar2/Bip is found together with recombinant IP in the culture medium where full-length Kar2/Bip accumulates in addition to large amounts of truncated Kar2/Bip. Thus, for judging UPR activating properties of the produced protein it is important to additionally analyze the medium not only for intact Kar2/Bip but also for truncated versions of this UPR reporter protein.