Figure 7
Gel mobility-shift assays of His 10 -SgcR3 with sgcR1R2 promoter region. A, Purification of recombinant SgcR3 after overexpression as a fusion protein with an N-terminal His10-tag in E. coli BL21(DE3). Lanes 1, 2, 3 and 4 contain samples of the insoluble lysate of induced cells, the soluble lysate of induced cells, flow through from the column, combined column washes respectively. Lanes 5–9 contain samples of eluates 1–5 eluted by buffer containing 500 mM imidazole. His10-SgcR3 protein from the eluate 5 was used in EMSA analysis. The molecular masses (kDa) of the protein markers (TransGen Biotech, Beijing, CN) are indicated. B, EMSA analysis of His10-SgcR3 with upstream region of sgcA1, sgcB1, sgcC1, sgcD2, sgcK, cagA, sgcR3 and sgcR1R2. Each of the lanes contains 20 fmol of fluorescently labeled promoter region DNA fragment. Lanes 2 also contain 13.5 pmol of purified recombinant His10-SgcR3 protein. C, EMSA analysis of His10-SgcR3 with sgcR1R2 promoter region. Each of the lanes contains 20 fmol of fluorescently labeled sgcR1R2 promoter region DNA fragment. Lanes 2–6 also contain 0.5 pmol, 3.12 pmol, 6.25 pmol, 13.5 pmol and 27 pmol of purified recombinant His10-SgcR3 protein, respectively. Lane 7 contains 6.25 pmol His10-SgcR3 and 200 fold excess unlabeled sgcR1R2 promoter region DNA fragment.