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 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.

Integrated molecular and bioprocess engineering for bacterially produced immunogenic modular virus-like particle vaccine displaying 18 kDa rotavirus antigen.

A high global burden of rotavirus disease and the unresolved challenges with the marketed rotavirus vaccines, particularly in the developing world, have ignited efforts to develop virus-like particle (VLP) vaccines for rotavirus. While rotavirus-like particles comprising multiple viral proteins can be difficult to process, modular VLPs presenting rotavirus antigenic modules are promising alternatives in reducing process complexity and cost. In this study, integrated molecular and bioprocess engineering approaches were used to simplify the production of modular murine polyomavirus capsomeres and VLPs presenting a rotavirus 18 kDa VP8* antigen. A single construct was generated for dual expression of non-tagged murine polyomavirus capsid protein VP1 and modular VP1 inserted with VP8*, for co-expression in Escherichia coli. Co-expressed proteins assembled into pentameric capsomeres in E. coli. A selective salting-out precipitation and a polishing size exclusion chromatography step allowed the recovery of stable modular capsomeres from cell lysates at high purity, and modular capsomeres were successfully translated into modular VLPs when assembled in vitro. Immunogenicity study in mice showed that modular capsomeres and VLPs induced high levels of VP8*-specific antibodies. Our results demonstrate that a multipronged synthetic biology approach combining molecular and bioprocess engineering enabled simple and low-cost production of highly immunogenic modular capsomeres and VLPs presenting conformational VP8* antigenic modules. This strategy potentially provides a cost-effective production route for modular capsomere and VLP vaccines against rotavirus, highly suitable to manufacturing economics for the developing world. Biotechnol. Bioeng. 2017;114: 397-406. © 2016 Wiley Periodicals, Inc.