10.22376/ijpbs.2019.10.1.p1-12 Volume 4 Issue 4 2013 (October - December) Recombinant human erythropoietin (EPO) is a glycoprotein produced as a therapeutic agent for the treatment of anemia associated with severe kidney damage. The demand of this protein currently met by recombinant expression in mammalian cells. lessThan i greaterThan Pichia pastoris lessThan /i greaterThan has become popular yeast based protein production systems to substitute mammalian expression systems. In this study, recombinant human EPO (rhEPO) protein obtained by expressing the human lessThan i greaterThan epo lessThan /i greaterThan gene in methylotropic yeast lessThan i greaterThan P. pastoris, lessThan /i greaterThan strain X33. The human EPO cDNA was inserted into pPICZαB vector, under the control of AOX1 promoter, and fused with a polyhistidine tag and c-myc epitope. Several clones were screened by Western blot analysis using polyclonal anti-EPO antibodies. The highest expressing clone was selected for subsequent study. The recombinant human EPO (rHuEPO) protein produced was approximately 37 kDa in size. Analyses by SDS/PAGE, Western blot, deglycosylation and internal amino acid sequencing confirmed the authenticity of the expressed rHuEPO protein. SANTOSO A, RUBIYANA Y, WIJAYA SK, HERAWATI N, WARDIANA A AND NINGRUM RA Erythropoietin, EPO, Pichia pastoris, Yeasts 187-196