The Optimization Research Of Recombinant Human Serum Albumin In P. Pastoris

Human serum album(HSA)is about60percent of the total protein of the human plasma. It is largelyresponsible for maintaining the normal osmolarity of the blood stream and also have other function such asanticoagulation,clean the free radicals et al. HSA is widely used in anti-shock, treatment of burns,bleeding caused acute hypovolemia by surgery or accidental. At Present,on sale HSA prepared from humanplasma is limited in supply and there is the potential risk of contamination of the product by blood-drivedpathogens. It is a trend of replaced HSA to product rHSA by genetic engineering means. The problem ishow to raise the expression level and the purity of rhsa。Preliminary studies on the process of rHSAexpression with different combination of vector-strains and other factors in different scale were studied inthis thesis.Discused the different combination of vector-strains during rHSA expressing in this thesis, pPICZα isthe better expression vector than others; and X-33is the best host than GS115and SMD1168in theexpression level of rHSA. It means use X-33may raise up the expression level of rHSA.Discussed the different factors such as the different volume of liquid, the different rotate speed, thedifferent temperature, the different pH, the different concentration of methanol with different times, thedifferent during the expressing in1L flask. The best condition of ferment as: choose the100mL BMGYmedium in1L flask, the rotational speed is200rpm, the induced pH with5.5, the induced temperature is22℃, the induced concentration of methanol with1%of total volume, twice a day, the concentration ofphosphate is100mM/L, the concentration of ammonium sulfate is0.5%and during the optimizedprocession it can be up to0.5g/L after induced240h.Discussed the different factors such as the different basal salt medium, the different initial induced celldensity, the different dissolved oxygen(DO), the different induced pH, the different temperature, themethanol concentration, the extra carbon and oleic acid of fermentation medium during the expressing in3L fermenter. Finally determined the technology as: the inoculation is10%, the initial induced cell densityis400, maintain the DO at30%, the induced temperature is22℃, the induced pH with5.75, keep themethanol concentration at1%by methanol electrode, add2g/L glycerol per hour during the fermentation process, the concentration oleic acid is0.1%, On line control and data acquisition bythe fermenter control system software in the whole fermentation process. during theoptimized procession the highest expression of rHSA can be close to5g/L after induced288h.