Abstract
BackgroundPichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast.ResultsAddition of defined concentrations of Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka) or SB2121 (Struktol) to shake-flask cultures of P. pastoris increased the total amount of recombinant GFP in the culture medium (the total yield) and in the case of P2000, SB2121 and J673A almost doubled it. When normalized to the culture density, the GFP specific yield (μg OD595-1) was only increased for Antifoam A, Antifoam C and J673A. Whilst none of the antifoams affected the growth rate of the cells, addition of P2000 or SB2121 was found to increase culture density. There was no correlation between total yield, specific yield or specific growth rate and the volumetric oxygen mass transfer coefficient (kLa) in the presence of antifoam. Moreover, the antifoams did not affect the dissolved oxygen concentration of the cultures. A comparison of the amount of GFP retained in the cell by flow cytometry with that in the culture medium by fluorimetry suggested that addition of Antifoam A, Antifoam C or J673A increased the specific yield of GFP by increasing the proportion secreted into the medium.ConclusionsWe show that addition of a range of antifoaming agents to shake flask cultures of P. pastoris increases the total yield of the recombinant protein being produced. This is not only a simple method to increase the amount of protein in the culture, but our study also provides insight into how antifoams interact with microbial cell factories. Two mechanisms are apparent: one group of antifoams (Antifoam A, Antifoam C and J673A) increases the specific yield of GFP by increasing the total amount of protein produced and secreted per cell, whilst the second (P2000 or SB2121) increases the total yield by increasing the density of the culture.
Highlights
Pichia pastoris is a widely-used host for recombinant protein production
We looked at polypropylene glycol (PPG) P2000 that is analogous to previously-examined liquid single components of the PPG-type [11] as well as examples from other categories such as Antifoam A and Antifoam C, which are silicone polymers, SB2121, which is a polyalkylene glycol, and J673A, which is an alkoxylated fatty acid ester on a vegetable base and has not previously been documented in this context: for all antifoams examined, this was the first report of their effect on the yield of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of P. pastoris
Antifoam addition affects total GFP yield in shake flasks The total yield of GFP as a function of Antifoam A addition rose significantly at concentrations of 0.6% and above (Figure 1A) with no further increases above 1%
Summary
Pichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. The laboratory-scale production of recombinant proteins using P. pastoris requires that cells are cultured either in large shake flasks or in continuously stirred tank bioreactors. In these vessels, the formation of foam is an issue that requires intervention. While little information is routinely given about their composition, their specific antifoam properties have been thoroughly investigated These include their effects on foam height with time, their influence on the volumetric oxygen mass transfer coefficient (kLa) of the system, their gas hold-up characteristics and their globule size and distribution in relation to their action upon foams [3,5,7,8,9,10,11]. Such studies have been performed in various growth media in both the absence and presence of cultures of prokaryotic and eukaryotic microbes
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