检索规则说明:AND代表“并且”;OR代表“或者”;NOT代表“不包含”;(注意必须大写,运算符两边需空一格)
检 索 范 例 :范例一: (K=图书馆学 OR K=情报学) AND A=范并思 范例二:J=计算机应用与软件 AND (U=C++ OR U=Basic) NOT M=Visual
机构地区:[1]广东医学院附属医院病理科,广东湛江524001 [2]广东医学院附属医院儿科,广东湛江524001
出 处:《广东医学院学报》2015年第4期394-398,402,共6页Journal of Guangdong Medical College
基 金:广东省自然科学基金(No.S2013040012902);国家自然科学青年基金(No.31300602);广东医学院博士启动基金(No.XB1341)
摘 要:目的获得可用于蛋白晶体筛选的高纯度及构象均一的Nix蛋白。方法将Nix截断基因构建到原核载体并转化大肠杆菌。异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后通过GST柱亲和纯化目标蛋白。TEV酶切除GST标签后采用凝胶过滤层析进一步纯化。结果 Nix截断基因成功构建到p GEX-4T-2并诱导表达了目标蛋白。通过GST亲和层析得到了可溶性的携带GST标签的Nix(2∽135)蛋白。切除GST标签的Nix(2∽135)通过凝胶过滤层析得到了构象均一的单体蛋白。结论采用原核表达、亲和层析和凝胶过滤层析可获得纯度高且构象均一的单体Nix蛋白。Objective To obtain the highly purified and homogeneous Nix for protein crystal screening. Methods The truncated Nix genes were constructed into prokaryotic vector and then transformed into Escherichia coli. The fusion proteins were induced by isopropyl-β-D-1-thiogalactopyranoside(IPTG), and then purified by GST affinity chromatography. The target proteins were further purified by gel filtration chromatography after GST tag was removed by TEV protease. Results The truncated Nix gene were constructed into p GEX-4T-2 recombinants and target proteins were successfully expressed. The soluble GST-Nix(2-135) was obtained by GST affinity chromatography. The monomeric protein was harvested from Nix(2-135) without GST tag using gel filtration chromatography. Conclusion The highly purified and homogeneous Nix monomeric protein was obtained using prokaryotic expression, affinity chromatography and gel filtration chromatography.
关 键 词:线粒体自噬受体Nix 载体构建 亲和层析 凝胶过滤层析
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在链接到云南高校图书馆文献保障联盟下载...
云南高校图书馆联盟文献共享服务平台 版权所有©
您的IP:216.73.216.249