Purification of recombinant human granulocyte colony-stimulating factor from Pichia pastoris using two ninta chromatography methods

Abstract

Human granulocyte colony-stimulating factor (hG-CSF) is a glycoprotein that stimulates the production of mature neutrophil and enhances its survival, proliferation, differentiation, and neutrofil precursor function. This study was carried out to determine the purity of recombinant protein employing two purification methods using NiNTA with imidazole and with pH gradient (without imidazole). The synthetic gene (gcsf-cmyc) was cloned into secretive expression vector pPICZαA and methanol utilizing alcohol oxidase (AOX1) promoters before being expressed in Pichia pastoris SMD1168H strain. The recombinant protein was purified using NiNTA chromatography with imidazole and pH gradient. All samples were analyzed using SDS PAGE, followed with detection using coomasie blue. The molecular mass of recombinant hG-CSF expressed in P. pastoris was ∼23kD. The efficiency of hG-CSF purification using NiNTA with imidazole was ∼63%, while with pH gradient was ∼89%. Purification techniques use pH gradients gradients can be applied to avoid used of imidazole, so that it does not contaminate protein samples.