1475-2859-10-17[1].pdf (420.02 kB)
Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield
journal contribution
posted on 2011-06-30, 09:17 authored by Sarah J. Routledge, Christopher Hewitt, Nagamani Bora, Roslyn M. BillPichia 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. Addition 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. We 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.
History
School
- Aeronautical, Automotive, Chemical and Materials Engineering
Department
- Chemical Engineering
Citation
ROUTLEDGE, S.J. ... et al, 2011. Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield. Microbial Cell Factories 10 (17), pp. 1-11.Publisher
BioMed Central (© The author)Version
- VoR (Version of Record)
Publication date
2011Notes
© 2011 Routledge et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.ISSN
1475-2859Publisher version
Language
- en