Microalgae are increasingly being considered for recombinant protein production because of low cultivation costs, absence of endotoxins and insusceptibility to human infectious agents. Despite these advantages, the yield of recombinant protein produced in microalgae is still low compared to more established expression systems and optimization at the genetic and cultivation levels is required for this new system to be economically viable. This study investigates the effect of biphasic temperature regimes on the yield of recombinant human interferon alpha 2a (IFN-α2a), a therapeutic protein known for its anti-cancer and anti-viral properties, produced by the model green alga Chlamydomonas reinhardtii (Cr.IFN-α2a). Biphasic growth is commonly employed to increase recombinant protein production in mammalian cell lines used for commercial production of therapeutic proteins, with a lowering of the temperature resulting in higher yields. In this study, lowering the temperature from 25 °C to 15 °C in mid-exponential growth phase increased the accumulation of Cr.IFN-α2a by 3.3-fold while it slowed down the growth of the three C. reinhardtii transgenic lines tested. In contrast, a rise of temperature from 25 °C to 35 °C accelerated cell growth, while negatively impacting the production of Cr.IFN-α2a. After a two-step chromatography purification, the Cr.IFN-α2a produced was estimated to be 53% pure with a yield of 90 μg/L of culture. The amino acid sequence of Cr.IFN-α2a was confirmed by mass spectrometry. However, the anti-viral activity of Cr.IFN-α2a was found to be 10 times lower than the human IFN-α2a standard produced using E. coli when challenged in a cytopathic effect (CPE) assay, likely due to the formation of aggregates. While the molecular mechanisms driving the accumulation of Cr.IFN-α2a at lower temperature remains unclear, our results support that reducing the temperature at the peak of expression is a valid strategy to increase the yield of recombinant Cr.IFN-α2a in C. reinhardtii.

由于培养成本低，无内毒素和不易感染人类传染病，因此越来越多地考虑将微藻用于重组蛋白的生产。尽管具有这些优点，但与更成熟的表达系统相比，微藻中产生的重组蛋白的产量仍然较低，并且需要在遗传和培养水平上进行优化才能使该新系统在经济上可行。这项研究调查了双相温度对重组人干扰素α2a（IFN-α2a）产量的影响，重组人干扰素α2a是一种以抗癌和抗病毒特性闻名的治疗性蛋白质，由模型绿藻莱茵衣藻（Crlamydomonas reinhardtii）（Cr。 IFN-α2a）。双相生长通常用于增加用于商业生产治疗性蛋白质的哺乳动物细胞系中重组蛋白质的产生，同时降低温度导致更高的产量。在这项研究中，将中指数生长期的温度从25°C降低到15°C，可使Cr.IFN-α2a的积累增加3.3倍，同时减慢了三个莱茵衣藻的生长。测试了转基因品系。相反，温度从25°C升至35°C会加速细胞生长，同时对Cr.IFN-α2a的产生产生负面影响。经过两步色谱纯化后，估计生成的Cr.IFN-α2a纯度为53％，培养物产量为90μg/ L。通过质谱确认了Cr.IFN-α2a的氨基酸序列。但是，发现Cr.IFN-α2a的抗病毒活性比使用大肠杆菌生产的人IFN-α2a标准低10倍。在细胞病变效应（CPE）分析中受到挑战时，可能是由于聚集体的形成。虽然尚不清楚在较低温度下驱动Cr.IFN-α2a积累的分子机制，但我们的结果支持降低表达高峰时的温度是增加重组C.reinhardtii中Cr.IFN-α2a产量的有效策略。。