High Expression of Human Cathepsin S by Recombinant Pichia pastoris with Cod Skin as an Organic Co-Nitrogen Source

Abstract

Human cathepsin S production by recombinant <i>Pichia pastoris</i> using cod skin as the co-nitrogen source was investigated in this study. The addition of carbon sources of glycerol in the fed-batch phase and of methanol in the induction stage was also investigated. A new approach to the highly expression of human cathepsin S was developed using 90 g/L of cod skin (wet weight). After 24 h of the initial fermentation, 4% glycerol (v/v, glycerol/culture) was added once to enhance the cell density (OD₆₀₀) in the cultivation. Then, adding and maintaining methanol at 0.5% (v/v, methanol/cultivation) after about 48 h of fermentation achieved a high expression of human cathepsin S in a 5-L bioreactor. The results demonstrate that the maximum activity of human cathepsin S in the fermentation supernatant reached 7,152 U/L after 96 h of methanol induction. The methylotrophic yeast <i>P. pastoris</i> grown in the medium containing cod skin (90 g/L) as the co-nitrogen source provided a 21% higher cell density (OD₆₀₀) and 18.3% higher human cathepsin S yield than <i>P. pastoris</i> grown in BMGY medium. For the first time, human cathepsin S was successfully expressed by <i>P. pastoris</i> with<i></i> cod skin as the co-nitrogen source. The glycerol fed-batch controlling strategy and method of maintaining methanol at a constant concentration of 0.5% (v/v, methanol/cultivation) in the induction stage was efficient for <i>P. pastoris</i> growth and the expression of human cathepsin S.