Fermentation Production Of Recombinant Human Serum Albumin By Pichia Pastoris

Human serum album (HSA) is the major protein component of the human plasma, and is about 60 percent of the total protein of the human plasma. HSA is produced in the liver and is largely responsible for maintaining the normal osmolarity of the blood stream, but it also functions as a carrier for numerous small molecules. HSA plays an important role in clinical therapy of severe hypoalbuminemia or traumatic shock. The methylotrophic yeast, Pichia pastoris, with many advantages such as high expression, stable genetics and protein secretion, is being studied since it has been found to be a useful organism for efficiently expressing recombinant HSA. In this paper, we studied the high density cultivation of a combined strain of producing-rHSA Pichia pastoris and the separation of rHSA by ion exchange chromatography. The conditions of the growth phase were initially investigated. The optimal seed medium was YPD. The volume of medium was 50ml in a 500ml baffled flask. The concentration of glycerol was 4%, and (NH4)2SO4 was 10g/L. Add methanol to maintain induction without centrifuging after the growth phase.On the basis of the synthesized medium, the influences of(NH4)2SO4,PTM1,oleic acid,methanol and pH on the expression of recombinant human serum albumin were investigated. It was discovered that PTM1 significantly improved the expression of rHSA and the optimum concentration of PTM1 was 2-6ml/Lmethanol every 12 hours. The highest productivity of rHSA was obtained when the concentration of (NH4)2SO4 was 3g/L and the protein expression was depressed when the concentration was higher than 3g/L in the course of inducement. The optimum concentration of methanol was 10ml/L. The expression of rHSA was depressed when pH was lower than 6.0 and the optimum condition was pH6.0. Oleic acid improved the protein expression greatly and the optimum concentration was 0.05%. The above data could be used to direct the fed-batch fermentation.Different methanol feeding modes were evaluated in the rHSA production. It was difficult to avoid methanol excessive in the broth with the feeding strategy only based on DO level. On the other hand, the fluctuation in methanol concentration was observed with methanol feeding strategy by off-line gas chromatography. The supply of energy was improved by feeding glycerol at a limited rate as well as methanol in the induction phase. Adsorption and elution characteristics of human serum album and rHSA under different operation conditions, such as pH, ionic strength of buffer solution and concentration of feed were investigated. It was experimentally shown that high resolution and recovery of recombinant human serum album were obtained.