Purification of His-Tag proteins are based on the ability of six consecutive histidine residues on either the N-terminus or carboxyl terminus to bind to a resin containing nickel ions immobilized by covalently attached nitrilotriacetic acid (NTA).
Like any other large-scale purification of bacterial expressed proteins, the cells should be tested for expression of the proteins of interest and determined to see if the protein is soluble or insoluble. The following procedure will describe the basic method of binding and eluting His-tagged fusion proteins.
Preparation of NTA affinity column
NTA is usually cross-linked to Sepharose CL-6B, and has a light blue-green colour when charged with Ni 2+ and white when it is not charged. To charge the NTA resin, wash with 5 bed volumes of 100 mM NiSO4. 6H2O.
Buffers used on the NTA column should contain high salt in order to prevent unspecific electrostatic interactions.
Binding His-Tagged proteins to NTA resin
Usually, a charged column has a capacity of 5 to 10 mg histidine-tagged protein per mL of packed resin (refer to manufacture's information for specific details).
Load cell extract to charged column at a rate between 0.1 mL/min to 0.2 mL/min.
Save the flow through for SDS-PAGE.
Eluting His-Tagged proteins.
Proteins can be eluted from the resin by three methods.
1) Protease cleavage of the His-Tag. The protein of interest can be remove by using a specific cleavage site designed after or before the His-Tag. For example a thrombin site can be placed after the six His residue at the N-terminus of the protein. To elute the protein, elute the column with a buffer containing thrombin.
2) The second method uses imidazole to elute the His-Tag protein from the NTA resin. Imidazole concentration is usually increased from 0 M to 1 M, either by step increase or by using a gradient former. Typically, a Imidazole concentration of 100 mM to 200 mM is sufficient to elute 6 His proteins.
3) Another method involves using EDTA to remove the nickel from the column.
A combination of these elution steps can be used to get the most purified protein from the column. For example, a low concentration of imidazole can be used to remove any non-specific proteins bound to the NTA column. Then the protease can be used to remove the protein from the column.
The column can be totally eluted by using EDTA buffer. It will remove the Nickel on the column, changing the blue-green column to a white coloured column.