The Nickel-Nitrilo triacetic acid (Ni-NTA) coupled to Sepharose CL-6B from Qiagen offers a quick and efficient method of protein purification from small and large scale preparations obtained through variable expression sources like
E. coli, yeast, insect (baculoviruses) and mammalian expression systems. The purification technique is based on the strong affinity of resin-bound Ni for a 6 consecutive histidine (his) residues tag on recombinant proteins.
A few of the notable advantages of Ni-NTA resin are: i) flexibility of protein purification under both native and denaturing conditions, ii) in vitro refolding of recombinant protein, iii) one-step purification from cell lysates, resulting in greater than
90% pure protein under most conditions, iv) the resin can be recharged and thus, if maintained properly, can be used for a number of purifications, v) silica support allows minimum hydrophobic interactions, resulting in high purity with little polypeptide contamination.
The purified protein is suitable for a wide variety of studies such as structural and functional characterization of proteins, protein-protein interactions, protein-DNA interactions, immunization and crystallization.
To purify the recombinant protein under native conditions, lyse the cells in sodium phosphate buffer (pH 8.0) with NaCl, EDTA and protease inhibitors and centrifuge at high speed to obtain a clear cell lysate. The supernatant can then be mixed with the Ni-NTA slurry for 2 hr at 4ºC with gentle shaking/stirring or by passing it 3-4 times over a pre-packed Ni-NTA column (in other words batch or column binding). After washing with 10 column volumes, the bound protein can be eluted with an immidazole gradient (100-250 mM) or with 250 mM immidazole, a histidine analog. To increase the protein purity, the column can be washed with buffer containing 5-10 mM of immidazole with no loss of the desired protein.
The Ni-NTA resin can be used to purify inclusion bodies or mis-folded proteins as well. In these cases, the cells are lysed with 8 M urea or 6 M guanidine hydrochloride to expose the his tag; this method might also be explored for purification of recombinant proteins where the his tag is buried within the native 3-dimensional conformation. After lysis, the recombinant protein is bound to the column and refolded in vitro by gradual removal of denaturant followed by elution with immidazole. The solubilization at a high concentration of denaturant doesn’t in any way affect the binding affinity of Ni-NTA resin for the his tag or alter subsequent steps of protein purification. The refolded protein can be subsequently checked to determine the extent of folding. Perhaps, the only drawback is that in vitro refolding might not always yield a 100 % population of perfectly folded protein.
The best feature of the Ni-NTA resin is the flexibility of using strong denaturants, detergents and reagents for efficient solubilization and for the removal of nonspecific contaminants during subsequent purification steps of recombinant proteins. The resin is compatible with triton X-100, tween 20, chaps, sodium chloride, calcium chloride, and magnesium chloride.
The Ni-NTA affinity purification made the tedious process of protein purification simpler with minimum precautions while preparing and using the Ni-NTA column. I have used this resin for 6 years and have been absolutely satisfied with the results. I highly recommend this for all the researchers as an easy step to purify recombinant proteins.