TAILIEUCHUNG - analysis of genes and genomes phần 7

Thanh lọc của một protein, được gắn thẻ từ các tế bào E. coli được thể hiện trong hình . Các tế bào E. coli có chứa một vector biểu hiện cảm ứng được phát triển và gây ra để sản xuất các protein mục tiêu được gắn thẻ. | 278 PROTEIN PRODUCTION AND PURIFICATION 8 NH CO CH CH NH OC CH HN O C CH 2 CH C N O Ni2 -nitriloacetic acid OH CHo zCH NH CH CH XcH2 H2 CH2 CH2O Protein Spacer J Resin matrix Figure . The binding of proteins tagged with multiple histidine residues to Ni2 -NTA resin The purification of a his-tagged protein from E. coli cells is shown in Figure . E. coli cells containing an inducible expression vector were grown and induced to produce the tagged target protein. The cells were broken open and insoluble cell debris was removed by centrifugation. The supernatant from this process was applied to a Ni2 -NTA column. The column was washed with a low concentration 20 mM of imidazole which will compete with low-affinity histidine-column interactions to remove from the column any perhaps histidine-rich proteins that are non-specifically bound. Finally the tagged protein itself is removed from the column by increasing the concentration of imidazole to a high level 250 mM . This process results in the single-step purification of the tagged protein to yield a very pure almost homogenous sample. His-tagged proteins from any expression system including bacteria yeast baculovirus and mammalian cells can be purified to a high degree of homogeneity using this technique. Alternative elution conditions may also be used. For example lowering the pH from 8 to will alter the protonated state of the histidine residues and results in the dissociation of the protein from the metal complex. The tagged protein can also be removed by adding chelating agents such as EDTA to strip the nickel ions from the column and consequently remove the tagged protein. The small size of the histidine tag means that the tagged recombinant protein often behaves identically to its untagged parent. In some cases the tagged protein is actually found to be more biologically active than the untagged version of the same protein Janknecht et al. 1991 although this effect is likely to be due to the speed of .

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