Expression Of Human Recombinant ApoAI And D-amino Acid Oxidase Using Pichia Pastoris

Part one Apolipoprotein (ApoA) is an important liprotein of HDL (high-density lipoprotein) in which ApoAâ…  is a chief component known to have a wide range of physiology and pharmaceutical functions. Human blood has been the only source of this protein. However extracting ApoAâ…  from human serum has many questions such as low yield, complicated procedure, high cost and poor security. It is difficult to industralize for the target-drug. To obtain large quantity of the protein for its research and Application, some genetic expression systems were studied but could not get high expression. The P. pastoris expression system was first used for ApoAâ… expression by professor Da Xin Song. The gene fragment encoding human ApoAâ…  protein was obtained by PCR from DNA containing nature ApoAâ…  gene and was inserted into the secretion vector pPIC9K. Then the plasmid pPIC9k- ApoAâ… was transformed into P.pastoris GS115. 22 transformants with high G418 resistance (4mg/mL)were obtained from over 1000 transformants. This research was based on it. After the phentotype analysis, the expression ability of transformants were compared by growth in shaking flask and induction with methanol and SDS-PAGE analysis. Among them the 16th was the best and named AP16.The results of Western blot indicated that the ApoAâ…  from P. pastoris recombinant AP16 was similar to those of nature ApoAâ…  extracted from human serum. The emphasis in this pAper was to search the optimal conditions of growth and induction. The results showed that after growing in BMGY for 28 h, the recombinant AP16 was induced for 96h with 1% methanol in BMMY (pH 7.0-7.5) under the cells density at A600 =80( about wet weight 320g/L). The expression level reached 180 mg/L in 250 ml shaking flask, 9 times than that in Chinese hamster ovary cells(20mg/L) and 80% higher in Baculovirus-insect Cell System by Zhong Jiang. In 14L fermentation, after growing for 16h the cells reached high densities of about 150g/L and the expression level reached up 160 mg/mL the flask. The fermentation circle in fermentor was only 90h and was shorten at least 30h and was simple to control. Because of secret expression with high level the ApoAâ…  which accounted for 70% in the soluble protein was very easy to be purified. The success of high expression of ApoAâ…  shows the superiority of P.pastoris and will meet its large research, production and Application for the target-drug of liver.