摘要： N-糖基化修饰是毕赤酵母表达系统中最常见的对蛋白的一种表达后修饰方式，其糖基化修饰的差异可能会影响到酶在稳定性、活性和表达水平。毕赤酵母中的弹性蛋白酶的N-糖基化修饰位点序列为Asn-Xaa-Ser/Thr，而且，Asn-Xaa-Thr的糖基化效率比Asn-Xaa-Ser更高。本文对这两个序列在重组弹性蛋白酶（rPAE）中表达的作用进行了研究。在rPAE的N43，N212和N280这三个位点上，通过定点突变用Asn-Xaa-Thr是取代天然的Asn-Xaa-Ser序列，并且在rPAE的N36和N264位点的将这两个序列子形式引入。正如预期的那样，在N36，N43，N212和N280上，其糖基化水平显著地提高。结果验证了Asn-Xaa-Thr的糖基化水平比Asn-Xaa-Ser更高。58467

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 Pichia pastoris. Differences in glycosylation may affect stability, activity and expression level of the enzyme. N-glycosylation usually occurs at the Asn-Xaa-Ser/Thr sequon of glycoproteins in P. pastoris, however, Asn-Xaa-Thr is more efficiently glycosylated than Asn-Xaa-Ser. In this study, the role of the two sequons in the expression of recombinant elastase (rPAE) was investigated. At N43, N212, and N280 of rPAE, Asn-Xaa-Thr was substituted for the native Asn-Xaa-Ser sequon through site-directed mutagenesis, and the two sequon forms were introduced into rPAE at N36 and N264. As expected, substitution at N36, N43, N212, and N280 enhanced the degree of N-glycosylation. These results show that the tripeptide sequon Asn-Xaa-Thr is glycosylated more efficiently than Asn-Xaa-Ser.

Key words: N-glycosylation, Site-directed mutagenesis, Protein expression, Pichia pastoris 

引言： 4

1 实验材料及仪器 5

1.1 酶、菌株与质粒 5

1.2 试剂与溶液 6

1.3 实验仪器 6

2 实验方法 7

2.1 定点突变 8

2.2 目的基因的亚克隆 9

3 实验流程 10

3.1 基因定点突变 10

3.1.1 引物设计 10

3.1.2 原突变位点附近基因序列 10

3.1.3 引物序列 11

3.1.4 聚合酶链式反应 11

3.1.5 琼脂糖凝胶电泳 11

3.2 感受态制备和目的基因的转化 12

3.2.1 大肠杆菌感受态制备 12

3.2.2 目的基因的转化 12

3.2.3 提取质粒并测序 12

3.3 目的基因的亚克隆 12

3.3.1 乙醇沉淀DNA 12

3.3.2 酶切 13

3.3.3 目的基因的连接 13

3.3.4 再次转化筛选 13

3.4 诱导产酶 13

3.4.1 毕赤酵母电转化方法 13

3.4.2 菌体的准备 13

3.4.3 电击转化 14

3.4.4 诱导产酶培养 14

3.5 聚丙烯酰胺凝胶电泳