利用GAP启动子在毕赤酵母中表达与纯化GLP-1类似物  

Constituted expression and purification of glucagon-like peptide-1 analogue in Pichia pastoris using GAP promoter

作　　者：钱凯[1] 张晶晶 吴素平 蔡燕飞 陈蕴 金坚 

机构地区：[1]江南大学生物工程学院,无锡214122 [2]江南大学药学院,无锡214122

出　　处：《中国生物工程杂志》2015年第5期66-73,共8页China Biotechnology

基　　金：国家"863"计划(2014AA021003);中科院先导计划(XDA01040202)资助项目

摘　　要：胰高血糖素样肽-1(GLP-1)能提高II型糖尿病患者β胰腺细胞的胰岛素分泌量并能促进β胰腺细胞增殖,是潜在的糖尿病治疗药物。设计一种GLP-1类似物AGGH,即GLP-1(A2G)的二联体与人血清白蛋白(HSA)的N端连接,并在融合蛋白GGH前添加一个丙氨酸(A)。PCR获得融合基因aggh,将融合基因连接到p GAPZαA质粒中。在酵母中利用甘油醛三磷酸脱氢酶(GAP)启动子组成型表达外源蛋白AGGH。研究结果显示:筛选获得表达重组菌株,基因组PCR和western-blot验证正确;以葡萄糖为最优碳源培养下,表达量达到68 mg/L;5 L发酵罐中,发酵52 h蛋白产量最高达238 mg/L。蛋白经四步纯化后,获得纯度为95.8%的AGGH融合蛋白。与利用醇氧化酶1(AOX1)启动子表达的AGGH比较,发现两者产量和活性没有明显差异。但是,GAP启动子表达获得AGGH融合蛋白更加方便,且发酵时间减少了27.8%(20 h)。Glucagon-like peptide-1 （GLP-1） is a potential therapeutic drug for type II diabetes, mainly because of the stimulatory effect on insulin secretion under condition of high blood glucose. We used PCR to obtain a recombination gene AGGH, two GLP-1 （GLP-1A2G） mutants were connected in series and then fused to the N terminal of human serum albumin, and alanine was added at the N terminal of GGH. The fusion gene was inserted into pGAPZaA plasmid and expressed by the glyceraldehyde-3-phosphate dehydrogenase （GAP） promoter. The engineered strain was constructed and the recombinant P. pastoris successfully expressed the fusion protein AGGH. The yield of AGGH reached 68 mg/L after 72 h fermentation in a flask, using glucose as the optimal carbon source. Fed-batch fermentation was investigated in a 5 L bioreaetor, and the expression level of GGH reached 238 mg/L in 52 h. The fusion protein AGGH was purified in four steps, and the final purity was 95.8%. The bioactivity of AGGH was the same as that expressed in P. pastoris by the AOX1 promoter. This study described an efficient way to express AGGH fusion protein in P. pastoris using GAP promoter, fermentation was easy way to control without carbon source change than AOX1 promoter-controlled GGH expression and fermentation time was 20 h less than AOX1 promoter-controlled GGH expression.

关 键 词：毕赤酵母 GLP-1类似物 GAP启动子 

分 类 号：Q819[生物学—生物工程]