β-1，3-葡聚糖酶LamC的结晶条件筛选
毕业论文关键词：β-1,3-葡聚糖酶 表达纯化 蛋白质结晶
Screening of Crystallization Conditions of β-1,3-Glucanase LamC
Abstract：β-1,3-glucanase LamC from myxobacteria has high catalytic efficiency and special substrate specifity. It is amultifunctional enzyme with a single catalytic domain in C-terminal and a fascin-like domain in N-terminal.The function of fascin-like domainin the entire glycoside hydrolase family is currently unknown. X - ray crystal diffraction is one of the main methods to study the structure and function of proteins[1], and the premise is to obtain high quality protein crystals. In this paper, the Origami 2（DE3) harboring constructed expression vector pET32a-lamC were induced to express LamC. The protein was purified by nickel column affinity chromatography, dialysis and concentration. The purity of protein was detected through SDS-PAGE. Initial conditions were identified from the Hampton screen kits using sitting drop vapor diffusion. After optimization with the addictive screen kit, the LamC crystals were grown in 0.15 M Potassium bromide, 30% w/v Polyethylene glycol monomethyl ether 2000, 0.01 M GSH and 0.01 M GSSG at 5 mg/mL and 4°C, which laid the foundation for X - ray diffraction.
Key words: β-1,3-glucanase, expression and purification, crystallization
目录
摘要1
关键词1
Abstract1
Key words 1
1. 引言1
2. 材料与方法2
2.1菌株2
2.2培养基与溶液2
2.3实验器材3
2.4重组酶LamC的诱导表达3
2.5重组酶LamC的纯化3
2.6 重组酶LamC的SDS-PAGE检测 3
2.7 重组酶LamC的透析浓缩 4
2.8重组酶LamC浓度检测4
2.9重组酶LamC的结晶 5
3.结果与分析 6
3.1蛋白质纯化及纯度检测 6
3.2蛋白质浓度检测 6
3.3蛋白质结晶条件的初筛7
3.4 蛋白结晶条件的优化 8
4. 讨论 8
致谢 9
参考文献9
β-1，3-葡聚糖酶LamC的结晶条件筛选
1. 引言
β-1,3-葡聚糖酶在植物保护、饲料、酿酒、食品和医药工业等领域具有广泛的应用前景[2][3]。课题组前期克隆获得了新型β-1,3-葡聚糖酶LamC，LamC具有高效的催化效率和特殊的底物选择性。已报道的多功能糖苷水解酶大多由多个催化结构域组成，不同的催化活性由不同的活性中心完成，另一些含有一个催化结构域和一个或多个底物结合域。LamC由N端Fascin结构域和C端GH16催化结构域组成，目前Fascin结构域在整个糖苷水解酶家族中的功能未知，同一催化结构域水解多种底物的催化机理也有待探究。
研究蛋白质的结构对蛋白功能及其机制研究具有重要的意义，结晶是目前研究大分子结构的重要手段之一[4][5]。目前为止，已解析的GH16家族β-1,3-葡聚糖酶晶体结构有8个，其中文献报道的结构有5个，但LamC催化结构域序列与它们的序列一致性为24.5-34.5%，表明LamC结构具有新颖性。本课题拟通过蛋白质表达纯化，结晶条件筛选等获得可供X衍射的高质量晶体，为LamC的结构解析和催化机制研究奠定基础。