猪胃蛋白酶原A在毕赤酵母中的表达

Expression and Condition Optimization of Pepsinogen A in Pichia Pastoris

利用已有的猪胃蛋白酶原AcDNA,构建毕赤酵母pPIC3.5K-pA胞内表达载体.经纤维蛋白消化试验和干酪素琼脂平板法筛选,获得两株高表达的重组子,研究它们在不同pH值、不同诱导时间、不同起始浓度和不同甲醇诱导浓度等条件下表达水平的高低.结果表明:在pH值为7、诱导72h、起始诱导浓度的OD600为2、甲醇诱导浓度为1%时,重组胃蛋白酶活力最高,达到6.46U/mL.

Based on the cloning and sequence analysis ofpepsinogen A from the Hubei swine, constructed plasmid ofpPIC3.5K-pA in Pichiapastoris. There were screened two over-expression ofrecombinants by the experiment of hemaleucin digestion and the method of casein agar plate, and studied the effect of different pH, different induction time, different induction concentration and methanol induction concentration on the expression yield. The results indicated that, the activity of recombinant pepsin was able to reach 6.46 U/mL, under the conditions that the pH value was 7, induct 72 h, the induction concentration OD600 was 2, the methanol induction concentration was 1%.