Seasonal Weather Changes Affect the Yield and Quality of Recombinant Proteins Produced in Transgenic Tobacco Plants in a Greenhouse Setting.

Matthias Knödler,Jessica Emonts,Clemens Rühl,Johannes Felix Buyel

doi:10.3389/fpls.2019.01245

Abstract

Transgenic plants have the potential to produce recombinant proteins on an agricultural scale, with yields of several tons per year. The cost-effectiveness of transgenic plants increases if simple cultivation facilities such as greenhouses can be used for production. In such a setting, we expressed a novel affinity ligand based on the fluorescent protein DsRed, which we used as a carrier for the linear epitope ELDKWA from the HIV-neutralizing antibody 2F5. The DsRed-2F5-epitope (DFE) fusion protein was produced in 12 consecutive batches of transgenic tobacco (Nicotiana tabacum) plants over the course of 2 years and was purified using a combination of blanching and immobilized metal-ion affinity chromatography (IMAC). The average purity after IMAC was 57 ± 26% (n = 24) in terms of total soluble protein, but the average yield of pure DFE (12 mg kg−1) showed substantial variation (± 97 mg kg−1, n = 24) which correlated with seasonal changes. Specifically, we found that temperature peaks (>28°C) and intense illuminance (>45 klx h−1) were associated with lower DFE yields after purification, reflecting the loss of the epitope-containing C-terminus in up to 90% of the product. Whereas the weather factors were of limited use to predict product yields of individual harvests conducted for each batch (spaced by 1 week), the average batch yields were well approximated by simple linear regression models using two independent variables for prediction (illuminance and plant age). Interestingly, accumulation levels determined by fluorescence analysis were not affected by weather conditions but positively correlated with plant age, suggesting that the product was still expressed at high levels, but the extreme conditions affected its stability, albeit still preserving the fluorophore function. The efficient production of intact recombinant proteins in plants may therefore require adequate climate control and shading in greenhouses or even cultivation in fully controlled indoor farms.

Highlights

Plants have been developed as expression systems for the production of recombinant proteins including biopharmaceuticals (Hiatt et al, 1989), some of which are entering clinical trials (Ma et al, 2015), and a few are already on the market (Mor, 2015)
We did not observe any correlation between biomass and recombinant protein yields, but we found that the recovery and yield of intact full-length DFE after immobilized metal-ion affinity chromatography (IMAC) purification varied substantially—for example, falling within the range 3.9–30.0 mg kg−1 in the course of 1 year (Figures 2 and S1B–D)
Correlations (|r| > 0.70) were observed between protease activity and internal relative humidity, as well as Temp≥28 and illuminance above a threshold of 45 klx (Ill≥45),Av.tot. (Table S2), which is consistent with the reported heat-induced induction of plant protease activity (Bita and Gerats, 2013). These results suggested that, like the DFE yield, Total soluble protein (TSP) was affected by the weather throughout cultivation but especially during sprouting, whereas protease activity was more sensitive to weather influences at the end of the cultivation and shortly before harvest

Summary

Introduction

Plants have been developed as expression systems for the production of recombinant proteins including biopharmaceuticals (Hiatt et al, 1989), some of which are entering clinical trials (Ma et al, 2015), and a few are already on the market (Mor, 2015). The risk of contamination is elevated by the abundance of pathogens, animals, and agrichemicals in the open field, so fully contained facilities have been designed that allow the controlled cultivation of plants on a medium to large scale (Wirz et al, 2012; Holtz et al, 2015). Such facilities require substantial upfront investment and operate under complex and error-prone process control systems, which may offset some of the cost savings achieved by switching from mammalian cells to plants. Greenhouses offer an attractive compromise because they achieve sufficient containment with only moderate infrastructure costs, as shown by the use of greenhouse facilities to cultivate plants expressing a monoclonal antibody that was purified and used in phase I clinical trials (Ma et al, 2015; Sack et al, 2015)

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Results

Conclusion