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.

Abstract

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 usingtwo-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.