针对层析介质的孔径分析,基于刚性球状分子进入柱状孔的假设,分别采用高斯正态分布和对数正态分布描述孔径分布,利用简化的单孔分配因子模型,建立了孔径分布函数和分配因子Kd的关联,通过体积排阻层析实验测定系列标准物的Kd,从而拟合得...针对层析介质的孔径分析,基于刚性球状分子进入柱状孔的假设,分别采用高斯正态分布和对数正态分布描述孔径分布,利用简化的单孔分配因子模型,建立了孔径分布函数和分配因子Kd的关联,通过体积排阻层析实验测定系列标准物的Kd,从而拟合得到孔径分布信息,建立了逆体积排阻层析法。以葡聚糖作为分子大小的标准物,测定了5种典型层析介质(SP Sepharose FAST FLOW、Q Sepharose FAST FLOW、ToyopearlDEAE-650M、Streamline DEAE和Sephadex G-150)的Kd,计算和比较了不同介质的孔径分布,分析了介质的可吸附孔表面积等结构参数,证实了逆体积排阻层析法分析层析介质孔径分布的可行性和实用性。展开更多
Immobilization metal affinity chromatography(IMAC)and size-exclusive chromatography(SEC)have been widely used in the purification of recombinant protein.In order to apply the column chromatography to the separation an...Immobilization metal affinity chromatography(IMAC)and size-exclusive chromatography(SEC)have been widely used in the purification of recombinant protein.In order to apply the column chromatography to the separation and purification of the gene recombinant with histidine-tags,the column chromatographic separation characteristics of N-terminal histidine-tagged(N-AxCeSD)and C-terminal histidine-tagged(C-AxCeSD)gene recombinant protein AxCeSD,one of the subunit involved in the cellulose synthesis in Acetobacter xylinum were studied.In the ring-shaped three-dimensional structure of AxCeSD,N-terminal histidine-tags were located in the inner of ring,while C-terminal histidine-tags were located in the outer.A higher imidazole concentration was necessary for eluting the C-AxCeSD from the IMAC column due to the C-terminal histidine-tags had stronger chelating interaction with the Ni2+ on the IMAC media.Moreover,the retention time for eluting C-AxCeSD from the same SEC gel column was shorter than that for N-AxCeSD,because the larger protein homolog was formed in the C-AxCeSD solution through the inter-molecular hydrogen bonds between the C-terminal histidine-tags.展开更多
选取碳末端富含酸性氨基酸的拟南芥SnRK2.6(sucrose non-fermenting1-related protein kinase 2.6)和人源PDI(protein disulfide isomerase),以及近球形蛋白拟南芥PYL10 (PYR like protein 10),分别将重复酸性氨基酸序列添加到SnRK2.6(1...选取碳末端富含酸性氨基酸的拟南芥SnRK2.6(sucrose non-fermenting1-related protein kinase 2.6)和人源PDI(protein disulfide isomerase),以及近球形蛋白拟南芥PYL10 (PYR like protein 10),分别将重复酸性氨基酸序列添加到SnRK2.6(1-332)、PDI(1-440)、PYL10碳末端,利用大肠杆菌BL21重组表达,经过亲和层析,离子交换层析和分子排阻层析进行纯化,综合利用分析超速离心技术,分子排阻层析技术以及多角度静态光散射技术,研究人为设计的多聚氨基酸末端对蛋白质分子排阻行为,聚合状态和其他水力学性质的影响。结果发现,多聚酸性氨基酸末端虽不影响蛋白质分子的聚合状态,但会明显减少分子排阻色谱中蛋白质的洗脱体积,影响蛋白质分子的斯托克斯半径和轴长比等水力学性质。展开更多
文摘针对层析介质的孔径分析,基于刚性球状分子进入柱状孔的假设,分别采用高斯正态分布和对数正态分布描述孔径分布,利用简化的单孔分配因子模型,建立了孔径分布函数和分配因子Kd的关联,通过体积排阻层析实验测定系列标准物的Kd,从而拟合得到孔径分布信息,建立了逆体积排阻层析法。以葡聚糖作为分子大小的标准物,测定了5种典型层析介质(SP Sepharose FAST FLOW、Q Sepharose FAST FLOW、ToyopearlDEAE-650M、Streamline DEAE和Sephadex G-150)的Kd,计算和比较了不同介质的孔径分布,分析了介质的可吸附孔表面积等结构参数,证实了逆体积排阻层析法分析层析介质孔径分布的可行性和实用性。
文摘Immobilization metal affinity chromatography(IMAC)and size-exclusive chromatography(SEC)have been widely used in the purification of recombinant protein.In order to apply the column chromatography to the separation and purification of the gene recombinant with histidine-tags,the column chromatographic separation characteristics of N-terminal histidine-tagged(N-AxCeSD)and C-terminal histidine-tagged(C-AxCeSD)gene recombinant protein AxCeSD,one of the subunit involved in the cellulose synthesis in Acetobacter xylinum were studied.In the ring-shaped three-dimensional structure of AxCeSD,N-terminal histidine-tags were located in the inner of ring,while C-terminal histidine-tags were located in the outer.A higher imidazole concentration was necessary for eluting the C-AxCeSD from the IMAC column due to the C-terminal histidine-tags had stronger chelating interaction with the Ni2+ on the IMAC media.Moreover,the retention time for eluting C-AxCeSD from the same SEC gel column was shorter than that for N-AxCeSD,because the larger protein homolog was formed in the C-AxCeSD solution through the inter-molecular hydrogen bonds between the C-terminal histidine-tags.
文摘选取碳末端富含酸性氨基酸的拟南芥SnRK2.6(sucrose non-fermenting1-related protein kinase 2.6)和人源PDI(protein disulfide isomerase),以及近球形蛋白拟南芥PYL10 (PYR like protein 10),分别将重复酸性氨基酸序列添加到SnRK2.6(1-332)、PDI(1-440)、PYL10碳末端,利用大肠杆菌BL21重组表达,经过亲和层析,离子交换层析和分子排阻层析进行纯化,综合利用分析超速离心技术,分子排阻层析技术以及多角度静态光散射技术,研究人为设计的多聚氨基酸末端对蛋白质分子排阻行为,聚合状态和其他水力学性质的影响。结果发现,多聚酸性氨基酸末端虽不影响蛋白质分子的聚合状态,但会明显减少分子排阻色谱中蛋白质的洗脱体积,影响蛋白质分子的斯托克斯半径和轴长比等水力学性质。
