Difference between revisions of "Part:BBa K1151000"

Revision as of 21:32, 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

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

We therefore decided to study the NikR protein: first, we focused on its characteristic to bind nickel. We have developed a protocol of incubation of the protein with nickel sulfate (stock: 10 ug/ul), in presence of an Incubation buffer (20 mM Tris pH 7,6, 100 mM NaCl). These are the samples we tested (each has a final volume of 100 ul):

1. 1,2 ul (1 ug) NikR + 0,3 ul Nickel sulfate + 98,5 ul Incubation buffer

2. 6,1 ul (5 ug) NikR + 1,5 ul Nickel sulfate + 92,4 ul Incubation buffer

3. 12,2 ul (10 ug) NikR + 3 ul Nickel sulfate + 84,8 ul Incubation buffer

The samples were put on wheel at 4°C overnight.

In order to eliminate the nickel eccess we proceeded with a molecular exclusion chromatography on Sephadex G-25 resin.

Figure 6: Sephadex columns.

In this way the protein with the nickel-binding sites saturated will be released first from the column, collecting the eluate easily. The nickel in excess will remain trapped in the pores of the resin.

ICP-AES assay

Inductively-Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) is a type of emission spectroscopy that uses the inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element. The intensity of this emission is indicative of the concentration of the element within the sample.

Sequence and Features