关键词:

组氨酸标记, 固定化金属亲和层析, 尺寸排阻层析, AxCeSD

Abstract:

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.

Key words:

组氨酸标记, 固定化金属亲和层析, 尺寸排阻层析, AxCeSD