title: High Expression of Methylotrophic Yeast-Derived Recombinant Human Erythropoietin in a pH-Controlled Batch System.
abstract: To accomplish the worldwide demand for recombinant human erythropoietin (rHuEpo) as a therapeutic, application of cost-efficient expression system of methylotrophic yeast Pichia pastoris (P. pastoris) rather than mammalian cells is indispensable. Herein, a report on high levels secreted-expression of Pichia-derived rHuEpo by batch fermentation in a pH stabilized format is presented. The full length cDNA of rHuEpo was inserted into pPICZαA vector under control of AOX1 promoter, downstream of the secretion-α-factor and electroporated into P. pastoris strain X33. The highest expression transformant was selected by screening among the colonies surviving high concentration of Zeocin (1.0 mg/ml), followed by comparative small scale expression analysis by ELISA. Stabilization of pH around 6.0 by adding phosphoric acid into the culture media during induction period, improved the yield of expression to 150 mg/l of the media. Single-step Nickel-affinity chromatography was employed for purification of rHuEpo-6xHis to 80% purity. Analyses by SDS-PAGE, Western blot and N-terminal protein sequencing confirmed the authenticity of the 33 kDa expressed rHuEpo with a native N-terminal indicating the proper cleavage of secretion-signal. Results of this study, further confirmed the possibility of employing methylotrophic yeast for scaled up production aims of rHuEpo as a cost-efficient expression system when provided evidence for higher expression yields through application of pH-controlled systems.
authors: Faculty of Pharmacy, Tehran University of Medical Science, Tehran, Iran ; Research and Production Plant, Pasteur Institute of Iran, Karaj, Iran.
authors: Ahmad
authors: A
authors: Maleki
authors: Farzin
authors: F
authors: Roohvand
authors: Hosnieh
authors: H
authors: Tajerzadeh
authors: Hossein
authors: H
authors: Khanahmad
authors: Maryam B
authors: MB
authors: Nobari
authors: Ahmad
authors: A
authors: Beiruti
authors: Abdolhossein Rouholamini
authors: AR
authors: Najafabadi
keywords: Erythropoietin; Fermentation; Pichia pastoris; Yeasts