摘要： N-糖基化修饰是毕赤酵母中最常见的一种蛋白翻译后修饰，有70-90％的N-糖基化修饰位点（Asn-Xaa-Ser/Thr）被修饰。这种修饰对蛋白在内质网中的折叠和高尔基体的转运是至关重要的，最终影响蛋白的表达水平。序列Asn-Xaa-Thr比Asn-Xaa-Ser的N-糖基化修饰效率要高，因此，对蛋白的表达具有不同的影响。本论文研究在弹性蛋白酶的N36、N43、N212、N264和N280位点这两者不同的N-糖基化修饰位点类型对其表达水平的影响。结果表明在N212和 N280位点，将Asn-Xaa-Thr替代 Asn-Xaa-Ser后，分别提高了弹性蛋白酶表达水平43％和25％。58466

毕业论文关键词：N-糖基化，定点突变，弹性蛋白酶，毕赤酵母

Abstract: N-glycosylation is one of the most common forms of protein post-translational modification in P. pastoris ; approximately 70‒90% of the asparagine (Asn, N) residues in potential N-glycosylation sites (Asn-Xaa-Ser/Thr) are N-glycosylated. The critical role of N-glycosylation in protein folding and trafficking through the ER and Golgi is well known. The N-glycosylation efficiency of Asn-Xaa-Thr exceeds that of Asn-Xaa-Ser; in fact, the former sequon is reported to be glycosylated two to three times more often than the latter sequon. The two forms of the N-glycosylation site in proteins may have differential effects on expression. The objective of this paper was to study the effect of substituting Thr for Ser in the N-glycosylation sequons (N36, N43, N212, N264 and N280) of rPAE on expression of the protein in P. pastoris. Our results indicated that the substitution of Asn-Xaa-Thr for Asn-Xaa-Ser at N212 and N280 increased the recombinant Pseudomonas aeruginosa elastase production by 43% and 25%, respectively.

Key words: N-glycosylation, Site-directed mutagenesis, Pseudomonas aeruginosa elastase, Pichia pastoris

目  录

引言： 5

1 实验材料及仪器 9

1.1 酶、菌株与质粒 9

1.2 试剂与溶液 9

1.3 实验仪器 9

2 实验方法 10

2.1 定点突变 11

2.2 目的基因的亚克隆 11

3 实验流程 12

3.1 基因定点突变 12

3.1.1 引物设计 12

3.1.2 原突变位点附近基因序列 12

3.1.3 引物序列 13

3.1.4 聚合酶链式反应 14

3.1.5 琼脂糖凝胶电泳 14

3.2 感受态制备和目的基因的转化 15

3.2.1 大肠杆菌感受态制备 15

3.2.2 目的基因的转化 15

3.2.3 提取质粒并测序 15

3.3 目的基因的亚克隆 15

3.3.1 乙醇沉淀DNA 15

3.3.2 酶切 16

3.3.3 目的基因的连接 16

3.3.4 再次转化筛选 16

3.4 诱导产酶 16

3.4.1 毕赤酵母电转化方法 16

3.4.2 菌体的准备 16

3.4.3 电击转化 16

3.4.4 诱导产酶培养