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

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.

Techno-economic process modelling and Monte Carlo simulation data of uncertainty quantification in field-grown plant-based manufacturing

“…3 shows the growth rate “ r ( T )” values for a range of temperature. It confirms previously reported optimal growth range (18.5–28.5 °C) [7] which corresponds to 291.5–301.5 K.…”

The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labelling for NMR spectroscopy

“…In the future, we will use mid-term and longterm cultures with more than 50% v/v D 2 O to increase the degree of 2 H labelling. Overall, our data show that plant cells offer a promising alternative to other eukaryotic systems for the high-level incorporation of deuterium in proteins, overcoming the toxicity issues affecting mammalian cells (Kushner et al, 1999). In addition to an estimation of the labelling degree, the mass spectrometry data can be used to assess the presence of post-translational modifications in plants versus E. coli.…”

“…stained host cell proteins (HCPs) and GB1) in a sample were used to estimate GB1 purity. Samples were also analysed by dot blot (Opdensteinen et al, 2021) using ImageJ (Schneider et al, 2012) for quantitative comparison to His 6 -tagged GB1 standards at concentrations of 0.0, 1.5, 3.5, 7.5, 10.0, 20.0, 40 and 70.0 mg/ L. DsRed fluorescence in the expression controls was quantified as previously described using 12 standards in the 0-225 mg/L range (Kn ödler et al, 2019).…”

Reducing water uptake into BY‐2 cells by systematically optimizing the cultivation parameters increases product yields achieved by transient expression in plant cell packs

“…[ 28 ] IgG1 and IgG3 antibodies were quantified by surface plasmon resonance (SPR) spectroscopy, using sensors coated with protein A (IgG1) or protein G (IgG3) and 2G12 as a standard, as previously described. [ 24 ] DsRed was quantified using eight standards in the 0 to 225 mg L ‐1 range as previously described [ 29 ] and the concentration of total soluble protein (TSP) was determined using eight dilutions of bovine serum albumin in the range 0 to 2000 mg L ‐1 as previously described. [ 30 ] Osmolality‐dependent DsRed expression was approximated using a log‐normal model (Equation 1): $$\begin{equation}y = {y_0} + \frac{A}{{\sqrt {2\pi \cdot w\cdot x} }}{e^{\frac{{ - {{\left[ {\ln \frac{x}{{{x_c}}}} \right]}^2}}}{{2{w^2}}}}}\end{equation}$$where y 0 is the minimal value ( y ‐offset) of the distribution (here, the minimal DsRed expression), x is the independent variable (here, the osmolality), x c is the center of the distribution, w is the standard deviation (width) of the distribution, and A is the area under the curve.…”