Construction of a Pichia pastoris strain efficiently producing recombinant human granulocyte-colony stimulating factor (rhG-CSF) and study of its biological activity on bone marrow cells

Non-glycosylated, recombinant human granulocyte colony-stimulating factor (rhG-CSF), produced by Escherichia coli (filgrastim, leukostim) is widely used to treat a number of serious human diseases and aids in the recovery post bone marrow transplantation. Although glycosylation is not required for the manifestation of the biological activity of G-CSF, a number of studies have shown that the carbohydrate residue significantly increases the physicochemical stability of the G-CSF molecule. Therefore, the aim of the present study was to design a Pichia pastoris strain capable of producing glycosylated rhG-CSF, and to study its effects on rat bone marrow cells. The nucleotide sequence of the rhG-CSF gene has been optimized for expression in P. pastoris , synthesized, cloned into the pPICZαA vector and expressed under the control of the AOX promoter in P. pastoris X33. One of the selected clones secreting rhG-CSF, produced 100–120 mg/l of rhG-CSF three days post-induction with methanol. The recombinant cytokine was purified using two-step, ion-exchange chromatography. The final yield of purified G-CSF was 35 mg/L of culture medium. The biological activity of rhG-CSF was examined in rat bone marrow cells. The P. pastoris strain was designed to produce relatively high levels of rhG-CSF. The rhG-CSF protein had a strong stimulating effect on the growth of rat bone marrow cells, which was comparable to that of the commercial drug leukostim, but showed a more persistent effect on granulocyte cells and monocyte sprouts, enabling the enhanced maintenance of the viability of the cells into the 4th day of incubation.