镍离子亲和层析介质的制备及其用于组氨酸标记蛋白质的纯化

以交联琼脂糖微球为基质,通过环氧氯丙烷活化,偶联亚胺二乙酸并螯合N i2+,制备得到一种镍离子亲和层析介质。结果发现,在强碱条件下环氧活化率达到了38.0μmol/mL,最终N i2+配基密度达到了30.9μmol/mL,偶联效率为81.3%。利用得到的镍离子亲和介质对基因工程大肠杆菌表达的组氨酸标记3-羟基丁酮还原酶进行了一步层析纯化,酶活回收率达到了58.8%,纯化倍数为2.1,蛋白纯度在85%左右,纯化效果与常用商品介质基本相当。

N-和C-末端组氨酸标记基因重组AxCeSD的柱层析分离特性

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.

Ni~Ⅱ-NTA修饰的银纳米粒/多孔硅芯片在线分离组氨酸标记蛋白和MALDI-TOF质谱检测(英文)

本文通过沉积在多孔硅表面的银纳米粒吸附对氨基苯硫酚和氨基的化学转化得到终端为NiII-Nα,Nα-二(羧甲基)-L-赖氨酸水合物-即NiⅡ-NTA体系的芯片。NiⅡ-NTA修饰的芯片被用于从高浓度的盐和助溶剂的缓冲体系中亲和捕获组氨酸标记的融合蛋白:thioredoxin-urodilatin和SUMO-hu-aprotinin,并进行在线的MALDI-TOF质谱检测,克服了MALDI-TOF质谱中直接点样污染物妨碍样品与基质共结晶的问题,避免了繁琐的离线样品预处理。芯片在线分离、纯化和MALDI-TOF质谱分析体系有望在复杂或原始体液的溶液中分析目标分子。