Bawa, Zharain, Routledge, Sarah J., Jamshad, Mohammed, Clare, Michelle, Sarkar, Debasmita, Dickerson, Ian, Ganzlin, Markus, Poyner, David R. and Bill, Roslyn M. (2014). Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks. Microbial Cell Factories, 13 (1),
Abstract
Background - Pichia 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). Results - Functional 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. Conclusions - The 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.
Publication DOI: | https://doi.org/10.1186/s12934-014-0127-y |
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Divisions: | College of Health & Life Sciences > School of Biosciences College of Health & Life Sciences > Chronic and Communicable Conditions College of Health & Life Sciences College of Health & Life Sciences > Aston Pharmacy School College of Health & Life Sciences > School of Biosciences > Cellular and Molecular Biomedicine |
Additional Information: | © 2014 Bawa et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Funding: BSRC CASE studentship with AstraZeneca Ltd, BBSRC grant BB/I019960/1, NIH grant DK52328 to ID. |
Uncontrolled Keywords: | Adenosine A receptor,Bioprocess,CD81,CD82,CGRP-RCP,Claudin-1,GFP,GPCR,HRP,Methanol induction,P. pastoris,Pre-induction expression,Biotechnology,Bioengineering,Applied Microbiology and Biotechnology |
Publication ISSN: | 1475-2859 |
Last Modified: | 05 Aug 2024 13:07 |
Date Deposited: | 19 Aug 2019 09:35 |
Full Text Link: |
http://www.micr ... ontent/13/1/127 |
Related URLs: |
http://www.scop ... tnerID=8YFLogxK
(Scopus URL) |
PURE Output Type: | Article |
Published Date: | 2014-09-04 |
Authors: |
Bawa, Zharain
Routledge, Sarah J. Jamshad, Mohammed Clare, Michelle Sarkar, Debasmita Dickerson, Ian Ganzlin, Markus Poyner, David R. ( ![]() Bill, Roslyn M. ( ![]() |