Cloning and expression of human granulocyte-macrophage colony stimulating factor (hGM-CSF) in Pichia pastoris

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a glycoprotein, belongs to the family of colony stimulating factors (CSFs) that regulate proliferation and differentiation of hematopoietic cells. Recombinant hGM-CSF (rhGM-CSF) is one of FDA-appoved, therapeutic recombinant proteins that have been successfully used to treat many diseases like neutropenia, leukemia, or in combination with chemical therapy, etc. In this study, we reported the production of rhGM-CSF in methylotrophic yeast Pichia pastoris through secretory expression using the inducible AOX1 promoter. The gene hgm-csf encoding for hGM-CSF comprising of 415 bps was isolated using PCR reaction with two primers hGM-F and hGM-R bearing XhoI and NotI restriction sites, respectively. After double digesting with these enzymes, the hgm-csf fragment was cloned into pPICZαA shuttle vector, between the XhoI and NotI sites. Recombinant vector containing the gene hgm-csf, termed pPICZαA/hGMCSF, under the control of promoter AOX has the ability to express hGM-CSF in P. pastoris. The accuracy of cloning strategy was confirmed by digestion with REs, PCR and sequencing. Sequencing alignment showed that the cloned gene completely homologous to those published on Genebank. SacI linearized pPICZαA/hGM-CSF was transformed into P. pastoris X33 strain to establish the recombinant P. pastoris X33::hgm-csf. In methanol-contained, inducing BMMY medium, the recombinant X33 cells expressed and secreted hGM-CSF into the supernatant. The secreted hGM-CSF migrated as a band at about 20 kDa, a diffuse band at the range of 29 to 35 kDa, indicating differentially glycosylated forms, and a band at 14,7kDa which is a nonglycosylated form on SDS-PAGE gel. This result was confirmed by Western blot with specific antibodies.