Seasonal Weather Changes Affect the Yield and Quality of Recombinant Proteins Produced in Transgenic Tobacco Plants in a Greenhouse Setting
Seasonal Weather Changes Affect the Yield and Quality of Recombinant Proteins Produced in Transgenic Tobacco Plants in a Greenhouse Setting
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DOI：
10.3389/fpls.2019.01245
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发表时间：
2019-10-08
影响因子：
5.6
通讯作者：
Buyel, Johannes Felix
中科院分区：
文献类型：
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作者：
Knoedler, Matthias;Ruehl, Clemens;Buyel, Johannes Felix
转基因植物具有在农业规模上生产重组蛋白的潜力，每年的产量可达几吨。如果温室等简单的栽培设施能够用于生产，转基因植物的成本效益就会增加。在这样的背景下，我们表达了一种基于荧光蛋白DsRed的新型亲和配体，并将其作为HIV中和抗体2F5的线性表位ELDKWa的载体。在2年的时间里，连续12批转基因烟草植株表达了DsRed-2F5表位(DfE)融合蛋白，并用漂烫和固定化金属离子亲和层析(IMAC)相结合的方法进行了纯化。经iMac处理后，其可溶性总蛋白的平均纯度为57+/-26%(n=24)，但纯DFE的平均产量(12 mg kg(-1))有较大的变化(+/-97 mg kg(-1)，n=24)，且与季节变化相关。具体地说，我们发现温度峰值(>；28℃)和强烈照度(>；45klx h(-1))与纯化后较低的DFE产率有关，反映了高达90%的产物中含有表位的C末端的丢失。虽然天气因素对预测每批(间隔1周)的单次收获的产品产量的作用有限，但使用两个独立预测变量(照度和植株年龄)的简单线性回归模型可以很好地近似平均批量产量。有趣的是，通过荧光分析确定的积累水平不受天气条件的影响，但与植物年龄呈正相关，表明该产物仍在高水平表达，但极端条件影响了其稳定性，尽管仍保留了荧光团的功能。因此，在植物中高效生产完整的重组蛋白可能需要适当的气候控制和温室遮荫，甚至需要在完全控制的室内农场种植。
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 degrees 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.