Abstract
We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5′ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5′ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5′ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of Agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at >20% total soluble protein, corresponding to 1.8 mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1 mg/g leaf fresh weight.
Highlights
Recombinant protein production systems have become an integral part of medicine, industry, and research
We show that optimizing the 5 untranslated regions (UTRs) and 3 transcription terminator region substantially enhances the production of green fluorescent protein (GFP), Norwalk virus capsid protein (NVCP), and the monoclonal antibody rituximab
We found that the 63nt AtPsaK 5 UTR produced intense green fluorescence, and gel quantification data indicate that GFP production was increased by >20% compared to the tobacco mosaic virus (TMV) 5 UTR (Figures 3A,B)
Summary
Recombinant protein production systems have become an integral part of medicine, industry, and research. Biopharmaceutical proteins, including monoclonal antibodies, enzymes, growth factors, and other biologics, are the largest and fastest growing sector of all pharmaceuticals (Butler and Meneses-Acosta, 2012). Most of these recombinant proteins are made with traditional bioreactors using mammalian, insect, or microbe cell cultures. Plant systems have been extensively explored as alternative expression systems that offer safety, cost-effectiveness, scalability (Huang et al, 2009; Thuenemann et al, 2013a; Klimyuk et al, 2014; Mortimer et al, 2015). The economic feasibility of plantbased systems is strongly yield dependent, and methods of increasing transgene expression are crucial for the success of plants as a recombinant protein production platform
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