Recombinant Human Serum Albumin Expressed In Yeast Pichia Pastoris

Human serum albumin (HSA) plays an important role in clinical therapy. At present, HSA prepared from human plasma is limited in supply and there is of the potential risk of contamination of the product by blood-drived pathogens. Pichia pastoris expression system has achieved much wider use in expressing the recombinant protein thanks to its many attractive merits. This dissertation was focused on expressing the recombinant human serum albumin (rHSA) in host of Pachia pastoris. Also the parameters of fermentation and the purification of rHSA were investigated.The secretion plasmid pPIC9K-hsa has been constructed from pPIC9K vector and cDNA of HSA. The pPIC9K-hsa then was linearized and integrated into AOX1 gene of Pichia pastoris chromosome by electroporation. Due to the marker gene HIS4, the transforments were incubated on MD/MM medium to select his+Muts phenotype. Based on this, we continued to screen out multicopy colonies by G418.Whereafter through baffled flask cultured in BMGY/BMMY medium, we found that the concentration of rHSA improved as the copy number increased. Among the screened out colonies, the GS115-rHSA-8 expressed the highest yield of rHSA. Lastly the rHSA immunity has been validated by western blotting.To enhance the expression of rHSA from GS115-rHSA-8, several parameters have been investigated. The primary results have been drawn as following: the 0.15g/L histidine could improve the rHSA yield 57.1%. The 0.10% oleic acid also could improve 43.4%. The 0.5% volume of methanol is effective in rHSA expression, but the protein expression has been inhibited when methanol concentration arrived at 1% V/V, especially at 2%. In addition, the adding of glycerol is useful to protein expression in induce phase, on the other side, the 0.2% (V/V) glycerol inhibited the expression. In induce phase, we found that when the ammonium sulfate concentration is 7g/L the rHSA expressed better than other concentration. To control the proteolytic degradation of rHSA, we have investigated the effect of pH, temperature and adding nitrogen resource on degradation. As a result, we found that pH 7.0 and addition of 1.5% YP(Yeast extract, 5g/L, Peptone, 10g/L) has been effective. Also, the 100μm PMSF had a good control of degradation.A simple and convenient purification procedure was also established. The supernatant was firstly heated at 60℃ for 8 hours and centrifugalized to get rid of the sediment. Then the heat-treated solution was dialyzed to exchange the 0.005M pH6.4 sodium phosphate buffer. Lastly, with the solution absorbed by DEAE Sephrose FF resin, the rHSA could be eluted by 0.005M pH6.4 sodium phosphate buffer with 0.10 mol/L and 0.20 mol/L NaCl gradient concentration. This present technological process may be beneficial to the purification of rHSA expressed by P. pastoris, and may be useful to the other protein expressed by P. pastoris.