Engineering the green algae Chlamydomonas incerta for recombinant protein production
Fig 2
C. incerta exhibits higher secretion and expression of mCherry compared to C. reinhardtii. A) mCherry fluorescence readings at excitation wavelength of 580/9 nm and emission wavelength of 610/20 nm for C. incerta (n = 84) and C. reinhardtii (n = 84) demonstrated a 3.5-fold higher fluorescence intensity for C. incerta than C reinhardtii (p-value < 2.2 x 10-16). Fluorescence readings were normalized with cell density using chlorophyll fluorescence. Threshold for positive expression (blue dashed line at 9.10 x 10-3 RFU for C. incerta and green dashed line at 0.580 RFU for C. reinhardtii) was defined as three standard deviations above the mean activity level observed in the wild types. B) Immunoblot analysis using an anti-RFP antibody indicated the expected band size of mCherry at approximately 30 kDa for both C. incerta and C. reinhardtii, with a 3.76-fold higher band intensity for C. incerta. C) Fluorescent microscopy images showed mCherry signals inside vesicles localized within the cell’s secretory pathway. D, E) Growth curves of the top-expressing clone indicated that mCherry secretion from the transgenic C. incerta line did not affect cell growth compared to transgenic C. reinhardtii lines and their respective wild types.