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摘要：本研究旨在使用原核表达体系E.coli.对新型N-糖酰胺酶PNGase Stca进行重组表达并研究其酶学性质。本研究成功将重组PNGase Stca质粒转入E.coli. BL21感受态细胞中进行表达，纯化得到了该目标蛋白，通过酶促反应实验发现该酶的最适pH为3.0，最适反应温度为37 ℃。该酶底物作用范围较广，相比于其他已知N-糖酰胺酶具有明显的优点，既可以作用于含高甘露糖型结构、也可以作用于含复杂型和杂合型结构的N-糖链，还可以作用于含α-1,3-核心岩藻糖结构的N-糖链。将PNGase Stca与PNGase A进行基因同源序列比对并完成PNGase Stca基因定点突变实验后发现，推测的6个关键氨基酸位点突变后酶仍然有较好的活性，证明这些位点的氨基酸对酶活性并不具有决定性作用。本研究结果可以为N-糖酰胺酶的作用机制和分子定向进化研究提供科学依据，为糖组学N-糖链研究提供一种更经济实用的新工具酶。25980
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毕业论文关键词：PNGase Stca；基因表达；蛋白纯化；酶学性质；定点突变
The cloning, expression and characterization of the novel N-Glycosidase PNGase Stca
Abstract: This research aims to express the novel N-glycosidase PNGase Stca in the Escherichia coli system and study its enzyme properties. In this research, recombinant plasmid containing PNGase Stca was successfully transformed into Escherichia coli BL21 cell and expressed. The target protein was purified. According to the results of enzyme catalysis, the optimum pH of PNGase Stca is 3.0 and its optimum temperature is 37 ℃. PNGase Stca has a wide spectrum of substrates and many advantages than other types of known PNGase. It can interact with N-linked glycans containing high-mannose, hybrid and complex structure as well as core α-1,3-fucose structure. Comparison of DNA homologous sequences between PNGase Stca and PNGase A were conducted, site-directed mutagenesis on PNGase Stca were made. Results show that after site-directed mutagenesis of the 6 speculated crucial amino acid sites, the mutant PNGase Stca remains to have a fine enzyme activity, indicating that these amino acid sites do not have decisive influences on enzyme activity. Experiment results mentioned above can make scientific foundations of the PNGase interaction mechanism and molecular directed evolution studies, providing a more economic and practical new type of tool enzyme for Glycomics studies.
Key words: PNGase Stca; gene expression; protein purification; enzyme properties; site-directed mutagenesis
目 录
摘要 1
关键词 1
Abstract 1
Key words 1
1 实验材料 2
1.1 菌种 2
1.2 主要试剂 2
1.3 主要仪器设备 2
2 实验方法 3
(一)PNGase Stca基因的合成与表达 3
2.1 重组质粒的转化 3
2.1.1 实验准备 3
2.1.2 实验步骤 3
2.2 重组PNGase Stca基因在E.Coli BL21中的表达 3
2.2.1 实验准备 3
2.2.2 实验步骤 3
(二)PNGase Stca基因表达产物的纯化 4
2.3 重组PNGase Stca基因表达产物的收集 4
2.3.1 实验准备 4
2.3.2 实验步骤 4
2.4 重组PNGase Stca基因表达产物的Ni-NTA亲和层析纯化 4
2.4.1 实验准备 4
2.4.2 实验步骤 4
2.5 重组PNGase Stca基因表达产物的SDS-PAGE分析 5
2.5.1 实验准备 5
2.5.2 实验步骤 5