Is Pichia pastoris a realistic platform for industrial production of recombinant human interferon gamma?

Razaghi, Ali, Tan, Emilyn, Lua, Linda H. L., Owens, Leigh, Karthikeyan, O. P. and Heimann, Kirsten (2017) Is Pichia pastoris a realistic platform for industrial production of recombinant human interferon gamma?. Biologicals, 45 52-60. doi:10.1016/j.biologicals.2016.09.015


Author Razaghi, Ali
Tan, Emilyn
Lua, Linda H. L.
Owens, Leigh
Karthikeyan, O. P.
Heimann, Kirsten
Title Is Pichia pastoris a realistic platform for industrial production of recombinant human interferon gamma?
Formatted title
Is Pichia pastoris a realistic platform for industrial production of recombinant human interferon gamma?
Journal name Biologicals   Check publisher's open access policy
ISSN 1095-8320
1045-1056
Publication date 2017-01-01
Year available 2016
Sub-type Article (original research)
DOI 10.1016/j.biologicals.2016.09.015
Open Access Status Not yet assessed
Volume 45
Start page 52
End page 60
Total pages 9
Place of publication London, United Kingdom
Publisher Academic Press
Language eng
Abstract Human interferon gamma (hIFN gamma) is an important cytokine in the innate and adaptive immune system, produced commercially in Escherichia coli. Efficient expression of hIFN gamma has been reported once for Pichia pastoris (Wang et al., 2014) - a proven heterologous expression system. This study investigated hIFN gamma expression in P. pastoris replicating the previous study and expanding by using four different strains (X33: wild type; GS115: HIS(-)Mut(+); KM71H: Arg(+), Mut-and CBS7435: Mut(S)) and three different vectors (pPICZ alpha A, pPIC9 and pPpT4 alpha S). In addition, the native sequence (NS) and two codon-optimised sequences (COSI and COS2) for P. pastoris were used. Methanol induction yielded no expression/secretion of hIFN gamma in X33, highest levels were recorded for CBS7435: Mut(S) (similar to 16 mu g. L-1). mRNA copy number calculations acquired from RT-qPCR for GS115-pPIC9-COS1 proved low abundance of mRNA. A 10-fold increase in expression of hIFN gamma was achieved by lowering the minimal free energy of the mRNA and 100-fold by Mut(S) phenotypes, substantially lower than reported by Wang et al. (2014). We conclude that commercial production of low cost, eukaryotic recombinant hIFN gamma is not an economically viable in P. pastoris. Further research is required to unravel the cause of low expression in P. pastoris to achieve economic viability. (C) 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Formatted abstract
Human interferon gamma (hIFNγ) is an important cytokine in the innate and adaptive immune system, produced commercially in Escherichia coli. Efficient expression of hIFNγ has been reported once for Pichia pastoris (Wang et al., 2014) - a proven heterologous expression system. This study investigated hIFNγ expression in P. pastoris replicating the previous study and expanding by using four different strains (X33: wild type; GS115: HIS-Mut+; KM71H: Arg+, Mut- and CBS7435: MutS) and three different vectors (pPICZαA, pPIC9 and pPpT4αS). In addition, the native sequence (NS) and two codon-optimised sequences (COS1 and COS2) for P. pastoris were used. Methanol induction yielded no expression/secretion of hIFNγ in X33, highest levels were recorded for CBS7435: MutS (∼16 μg. L-1). mRNA copy number calculations acquired from RT-qPCR for GS115-pPIC9-COS1 proved low abundance of mRNA. A 10-fold increase in expression of hIFNγ was achieved by lowering the minimal free energy of the mRNA and 100-fold by MutS phenotypes, substantially lower than reported by Wang et al. (2014). We conclude that commercial production of low cost, eukaryotic recombinant hIFNγ is not an economically viable in P. pastoris. Further research is required to unravel the cause of low expression in P. pastoris to achieve economic viability.
Keyword Biopharmaceuticals
Interferon gamma
Low-abundance RNA
Minimum free energy
Pichia pastoris
Protein expression
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID 2.3.4
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: HERDC Pre-Audit
Australian Institute for Bioengineering and Nanotechnology Publications
 
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