Effect of biphasic temperature regime on therapeutic recombinant protein production in the green alga Chlamydomonas reinhardtii

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-alpha 2a), a therapeutic protein known for its anti-cancer and anti-viral properties, produced by the model green alga Chlamydomonas reinhardtii (Cr.IFN-alpha 2a). 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 degrees C to 15 degrees C in mid-exponential growth phase increased the accumulation of Cr.IFN-alpha 2a 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 degrees C to 35 degrees C accelerated cell growth, while negatively impacting the production of Cr.IFN-alpha 2a. After a two-step chromatography purification, the Cr.IFN-alpha 2a produced was estimated to be 53% pure with a yield of 90 mu g/L of culture. The amino acid sequence of Cr.IFN-alpha 2a was confirmed by mass spectrometry. However, the anti-viral activity of Cr.IFN-alpha 2a was found to be 10 times lower than the human IFN-alpha 2a 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-alpha 2a 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-alpha 2a in C. reinhardtii.