Evaluation of pH/buffering conditions effect on the optimization of Recombinant Human Erythropoietin expression in the methylotrophic yeast, Pichia pastoris

Document Type : Original Article

Abstract

Expression of recombinant proteins and drugs in Pichia pastoris has been in development since the late 1980s and the number of recombinant proteins produced in P. pastoris has increased significantly in the past several years. Unlike bacteria, this strain is capable of producing complex proteins with post translational modifications such as correct folding, glycosylation, proteolytic maturation. Most importantly the strain can secrete proteins to very high levels under the control of an efficient and highly regulated promoter of the alcohol oxidase gene, AOX1. In the present study by inserting the recombinant human erythropoietin (rHuEpo) gene downstream of the highly inducible AOX1 promoter and the pre-pro α-factor signal sequence, we were able to efficiently produce rHuEpo protein. Subsequently, we evaluated the role of the pH of P. pastoris growth medium and different types of buffers effects used in the expression-specific medium during the process of rHuEpo expression to optimize the production of rHuEpo. Our result indicated that the concentration of rHuEPO produced in the medium containing MES buffer (pH 6.0, 100 mM) is significantly higher than that in other pH/buffering conditions (P-value < 0.05). MES buffer with pH of 6.0 and 100 mM final concentration in the expression-specific medium regulated the pH/buffering conditions throughout the expression time with the significant increase in the concentration of expressed rHuEpo in comparison to the standard buffering protocol suggested by Invitrogen.

Keywords

• Pichia pastoris
• Recombinant Human Erythropoietin (rHuEpo)
• pH/buffering conditions

---