Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks

Abstract

BackgroundPichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A2a adenosine receptor (hA2aR), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP).ResultsFunctional hA2aR was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA2aR and GFP were still produced in the pre-induction phases. Both hA2aR and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake-flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures.ConclusionsThe production of recombinant hA2aR, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0127-y) contains supplementary material, which is available to authorized users.

Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris

“…4d). The AOX1 promoter is not known to have leaky expression when grown in shake flasks (Aw and Polizzi 2016), even though this is not the case for bioreactors (Bawa et al 2014). Therefore, this implies that there may be other stresses on α2, M1 and M2, such as the onset of starvation.…”

The synthesis of recombinant membrane proteins in yeast for structural studies

“…This observation suggests that high recombinant protein yields in methanol-grown cells are due not just to promoter strength, but also to the global response of P. pastoris to growth on methanol [25]. However, P AOX1 -driven expression is leaky; the recent characterization of pre-induction expression under the control of P AOX1 [26] indicates that the uncoupling of growth and protein synthesis in P. pastoris cells has not yet been achieved. The response of a series of inducible S. cerevisiae promoters to different carbon sources has also been studied [22]; this type of careful analysis of promoter expression patterns now opens up opportunities for dynamic regulation of recombinant protein production in S. cerevisiae.…”

“…We previously demonstrated the major impact of the induction regime on the yield of secreted GFP from P. pastoris cultures, showing the importance of matching the composition of the methanol feedstock to the metabolic activity of the cells [86]. P AOX1 is induced on methanol; however, when glucose (which has been shown to repress AOX1 expression) was the pre-induction carbon source, the adenosine A 2A receptor and GFP were still produced in the pre-induction phases in bioreactor cultures [26]. This study also reveals that a range of recombinant membrane proteins can be detected in the pre-induction phases of P. pastoris cultures when grown in bioreactors, but not shake-flasks.…”

“…As discussed in section 3.1, when P. pastoris cells were cultured in methanol, the majority of the total mRNA pool was associated with two or more ribosomes per mRNA (and therefore designated as highly-translated). Methanol is used to induce protein production under the control of P AOX1 in this yeast, suggesting that high recombinant protein yields may be associated with the global response of P. pastoris to methanol as well as promoter activity [26].…”

Pichia pastoris as an expression host for membrane protein structural biology

“…The AOX1 promoter is repressed in the presence of glucose or glycerol but strongly upregulated in the presence of methanol, making it a relatively tightly controlled expression system. However, recent research has identified protein expression occurring during the pre-induction phase in cultures grown in bioreactors but not in shaker flasks indicating that the promoter is leaky under certain conditions [6].…”