Production Of Human Growth Hormone Of Recombinant Pichia Pastoris Fermentation Conditions

Production of recombinant human growth hormone(rhGH)by genetically engineered Pichia pastoris was studied in this paper.The purpose was to find out the optimal medium and induction conditions which were suitable for the recombinant Pichia pastoris.We got the ideal cultivation substratum and induction conditions in shake-flask culture by monofactorial analysis and orthogonal experiment,then scaled the result of shake-flask to 12.8L fermenter under the obtained conditions.The work of this paper opened a new high-performance way to obtain rhGH.The results are as the follows:1.We selected the BSM as high-density culture medium and meliorated it further:the optimal carbon and nitrogen source were selected and the results were analyzed by using SPSS,we found the effect of glycerol as carbon source was better than glucose,the effect of yeast extract as nitrogen source was much better than(NH₄)₂SO₄, then optimized the amount of each ingredient of BSM,the result were:Glycerol 30g/L,yeast extract 14g/L,K₂SO₄13g/L,MgSO₄·7H₂O11 g/L,CaSO₄0.3 g/L,PTM₁ 4.4 M1,0.1mol/L phosphate buffer saline(pH=6.5).The outcome showed the improved BSM was suitable for recombinant Pichia pastoris'growth.2.The culture conditions such as seed growth time,induction time,methanol concentration and pH were investigated,the optimum culture conditions include:the optimal seed growth time was 24h,induction time was 30h,methanol concentration was 2%,pH is 6.3～6.9.3.Then we scaled the result of shake-flask to 12.8L Bioengineering L1523 fermenter under the optimum conditions.The fermentation strategy was studied in the feed-batch fermentation meanwhile,the maximum yield of recombinant human growth hormone reached 0.54g/L,the OD₆₀₀reached 350.4.The recombinant Pichia pastoris secretory protein's characteristics were researched by SDS-PAGE electrophoresis,and the result showed that the product just was rhGH.