Expression of Mouse Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) in Pichia pastoris

Background and Objectives: Granulocyte-macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factors involved in proliferation and differentiation of bone marrow precursor cells into granulocytes, monocyte that used in treatment of patient with cancer, therefore, it was decided to express murine GM-CSF as a recombinant protein in Pichia pastoris (P. pastoris). Materials and Methods: The protein sequence of mouse GM-CSF was obtained from Uniprot database and ordered for synthesis after back translation and optimization of coding sequence. It was subsequently cloned into pPICZαA expression vector expressed in Pichia pastoris. Expression and activity of GM-CSF was confirmed by SDS-PAGE, dot blot, Western blotting in the culture supernatant. Biological activity of the product was confirmed by promoting FDC-P1 cell growth. Non-linear regression analysis was used for fitting a curve onto the cell proliferation data plotted against the growth factor dilutions and calculation of EC50 value. Results: Sequence optimization improved CAI from 0.66 in the native to 0.86 in the optimized GM-CSF sequence through replacing 24% of the nucleotides. In dot blot analysis, GM-CSF expression reached its peak on 5 days and started to decline after 7 days. The GM-CSF protein was appeared as bands with apparent molecular mass nearly 16-17 kDa. It was estimated that 18.5 μg mL–1 of active murine GM-CSF was secreted into the culture supernatant. Conclusion: The recombinant GM-CSF protein was expressed in Pichia pastoris yeast and was biologically active in the FDC-P1 cell line proliferation.