Difference between revisions of "Part:BBa K1151000"

Revision as of 11:13, 27 September 2013

Nickel-responsive pleiotropic regulator (HpNikR)

The HpNikR protein is a pleiotropic regulator from Helicobacter pylori. In presence of nickel it can acts as an activator or a repressor depending of the specific promoter that contains its operator site. It consists of two dimeric DNA binding domains separated by a tetrameric regulatory domain that binds nickel. This domain corresponds to the C-terminal regulatory domain which contains four nickel binding sites at the tetramer interface. Binding nickel, then a conformational change allows it to activate or repress trascription.

Usage and Biology

Figure 1: HpNikR nickel-binding domain (tetramer).

Figure 2: Proteic sequence analysis with Uniprot.

NikR expression using BL21 (DE3) cells

First we made ​​competent BL21 cells and we transformed it with the plasmid containing NikR. We then proceeded with the normal protocol of induction with IPTG.

Figure 3: PAGE of the first induction.

Cytosol/membrane separation by Zerial method

To confirm that NikR is a cytosolic protein (not expressed in multivesicular bodies, and then in membrane) we performed a separation membrane-cytosol (Zerial method) (sample: 2-hours induced cells).

Figure 4: PAGE of the separation.

NikR purification by Ni-NTA resin

His-tagged NikR (sample: 2-hours induced cells) can be purified by Ni-NTA resin, which has a high affinity for histidine residues.

Figure 5: Purification result (Eluate 1: 0,82 ug/ul).

NikR incubation with nickel sulfate and Sephadex molecular exclusion chromatography

Sequence and Features