黃鰭鯛生長激素在酵母菌上之表現

Abstract

本實驗將黃鰭鯛生長激素cDNA接在α-因數領導序到的啟動子之後，共同接在2u質體上，完成後此載體先用限制?剪切分析確定之，將其轉形到酵母菌中，由抗藥性的標誌確定轉形成功後，把轉形株培養在選擇性培養基中，培養三天然後抽取蛋白質產物，為瞭解蛋白質產物是來自細胞之那一部分，將分析樣本分成四部分，1.培養酵母菌後之培養液，2.細胞抽出液，3.triton處理細胞碎片，4.urea處理細胞碎片．把這些樣本以SDS-PAGE蛋白質電泳分析，結果培養酵母菌後之培養液在22kDa附近以銀染顯現一蛋白質條紋比控制組(只含pMA56/α)多出，其蛋白質經免疫化學染色亦為正反應但產量低．此載體穩定性低在第三天三十代時只剩10％的穩定度．?改善生產量曾嘗試各種培養方法，在固體培養方面有更好的產量.此蛋白質只在酵母菌培養液中測得，故大多分泌到體外．另外因其產量不穩定猜測生產過程生長激素易被蛋白質分解?分解，故曾利用個別的氨基酸加在培養基內抑制蛋白質分解?，目前有幾種氨基酸加在培養基中會促進酵母菌的生長，但是否有抑制效果需進一步分析． ?了做比較又構築一非分泌性載體，把α-因數領導序列的啟動子換成 glyceraldehyde-3-phosphate hydrogenase (GAP) promotor, 將該載體轉形到酵母菌，以同樣方法培養, 並分析蛋白產物亦被証實在細胞抽出液的樣本中用電泳分析在22kDa上有一條紋比控制組多出，且免疫化學染色亦?正反應．樣本來自細胞抽出的部分，故不會分泌到體外，此結果並可顯示該生長激素在酵母菌中是為水溶性．

In order to simplify the purification of fish GH, a secreation vector was constructed with a 2 um Ori derivative yeast plasmid containing an α-factor leader sequense and an ypGHcDNA of yellow porgy growth hormone, which was transformed into Saccharomyces cervisiae. Transforments were selected with trp marker. The recombinant proteins produced were analyzed by SDS polyacryamide gel electrophoresis (SDS-PAGE). A band in the 22 KD region was obserred an antiserum against ypGH. To determine the protein in the yeast cell fraction, the cell culture were fractionated into 4 fractions; 1.supernatant, 2.solube cell extract, 3.triton treated broken cells and 4.urea treated broken cells.The ypGH was detected only in supernatant, indicating that ypGH was secreated from the cell. The stability of pMAyp was poor; the plasmid containing cell population was reduced from 100% to 25% on the second day (after 10 yeast generations) and to about 10% on the third day (about 30 generations). is about 10%. To improve the production of ypGH differet cutivation conditions. were tested; the yield of ypGH in the solid medium is higher than that obtained from the liquid midium. Addition of amino acid, e.g. leu and glu to the cuture medium resulted in improved yeast cell growth and proliferation. Since the yield of ypGHcDNA was low in the secretion vector system, nonsecretion vector was also constructed for comparison. The glycealdehyde-3-phosphate dehydrogenase promoter was used, to replace the α—leader peptide sequence and the protein prodced were analysed with SDS-PAGE, A band of 22KD was detected. and identified by Immunoblot analysis. The protien was detected inside the cell; only in samples prepared from soluble cell extract.