For our larger protein constructs that contain fluorophores, adding a 6-residue histidine tag to either fluorophore tail may bind to nickel – using principles of affinity chromatography – will allow us to capture our proteins quickly and without disturbing native function. Ideally, we would only need this as one purification step after taking our cell lysate, centrifuging, and discarding the pellet. Histidine is an amino acid that will most readily associate with nickel, which is a transition metal. Nickel is immobilized on the chromatography column. Our protocol used here is adopted from Bornhorst et al. in the journal Methods of Enzymology.
Purification of Proteins Using Polyhistidine Affinity Tags Joshua A. Bornhorst and Joseph J. Falke. Principles and Techniques of Biochemistry and Molecular Biology (Seventh edition) By Keith Wilson and John Walker.
Many of the materials and buffers used may be purchased from a supplier. We would use a QIAgen 50% Ni2+-NTA (nickel-nitrilotriacetic acid) mixed with ice to bind the Histidine tag. Washing the resin with our supernatant and buffer would emit fractions of protein not of interest to us. Only after the removal of His-tag protein with imidazole (competes with histidine for nickel) that fractions containing our purified protein would be eluted.
1 mL fractions can be run on an SDS-PAGE at 300 volts to ensure there is only one purified protein in the fraction. Since all our proteins are monomers, we can assume that only one band will show on the gel (apart from the MW standard). MW size may be estimated using software tools such as PyMOL for reference.
TEV-Protease (Tobacco Etch Virus) Technique
The TEV protease-technique is necessary when expressing our very small proteins – the coiled-coils for example that at only about 3kDA would be very difficult for E.coli to express on their own. TEV-protease targets a sequence most commonly ‘ENLYFQS’ with its catalytic triad (10). Expressing the coiled-coil with a His tagged GFP will allow us to isolate and then cleave it. Therefore, our construct would be; N – His-tag – GFP – ENLYFGQ- coil – C. Using 50 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, and 1mM DTT will function as a reaction buffer for the isolated protein after nickel chromatography. Typical reaction duration would be overnight. A good rule of thumb regarding how much TEV-protease to use is 1 OD at 280 nm of protease per 100 OD at 280 nm of substrate protein. Note that nm of substrate protein. Note that using the TEV-protease occurs after full expression and purification. An SDS -PAGE should be run again after cleavage to ensure that only the small coil is remaining in the fraction.
For more information about purifying our parts check out our protocol section – and consider reading the protocol on expression and purification of our biosensor protein constructs.