Is Pichia pastoris a realistic platform for industrial production of recombinant human interferon gamma?
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 MutSphenotypes, 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.
College of Science and Engineering, James Cook University, Townsville 4811, Qld, Australia; Centre for Biodiscovery and Molecular Development of Therapeutics, James Cook University, Townsville 4811, Qld, Australia.
Ali
A
Razaghi
The University of Queensland, Protein Expression Facility, Brisbane 4072, Qld, Australia.
Emilyn
E
Tan
The University of Queensland, Protein Expression Facility, Brisbane 4072, Qld, Australia.
Linda H L
LHL
Lua
College of Public Health, Medical and Veterinary Science, James Cook University, Townsville 4811, Qld, Australia.
Leigh
L
Owens
Department of Biology, Hong Kong Baptist University, Hong Kong.
O P
OP
Karthikeyan
College of Science and Engineering, James Cook University, Townsville 4811, Qld, Australia; Centre for Biodiscovery and Molecular Development of Therapeutics, James Cook University, Townsville 4811, Qld, Australia. Electronic address: Kirsten.heimann@jcu.edu.au.
Kirsten
K
Heimann
D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
C573415:IFNG protein, human; D011994:Recombinant Proteins; D007371:Interferon-gamma
Biopharmaceuticals; Interferon gamma; Low-abundance RNA; Minimum free energy; Pichia pastoris; Protein expression