It’s probably a safe bet that most folks involved in biomedical research are well aware of the benefits of expressing recombinant proteins as fusions with epitope tags. While there are many different epitope tags from which to choose, the (His)6 epitope is one of the smallest and least likely to cause problems downstream. In addition, immobilized metal affinity chromatography (IMAC), the method of choice for His-tagged protein affinity purification, is robust and simple to perform. I recently had the opportunity to try a product that makes purification of His-tagged proteins even simpler. The product is Ni Sepharose 6 Fast Flow from GE Healthcare (formerly Amersham Biosciences).
Ni Sepharose 6 Fast Flow consists of a metal chelating moiety coupled to highly cross-linked 90 um agarose beads. While this is not much different from other IMAC resins, the chelating agent is novel. Most IMAC resins employ either nitriloacetic acid (NTA) or iminodiacetic acid (IDA); Ni Sepharose 6 Fast Flow contains a proprietary chelator and comes pre-charged with nickel. The resin is reported to have a protein binding capacity of up to 40 mg His-tagged protein/ml resin and exhibits minimal leaching of Ni+ during normal usage. In addition, it is stable in the presence of many common buffer additives, including high levels of reducing agents, denaturing agents, and detergents. If needed, the resin can be stripped of metal ions using EDTA and recharged with Ni+ or your metal ion of choice. Uncharged Sepharose Fast Flow is also available for optimizing purification schemes with different metal ions. An additional difference worth mentioning is that the company recommends including 20-40 mM imidazole in the binding and wash buffers. This is distinct from other IMAC resins where binding buffers often are imidazole-free and wash buffers are recommended to contain 5-10 mM imidazole.
I used Ni Sepharose 6 Fast Flow in a spin-column format to purify a peptide/bacterial alkaline phosphatase (BAP) fusion protein. I subcloned the DNA fragment coding for the 16-amino acid residue peptide from an M-13 phage vector into the BAP vector (BAP protein also carries a His-tag). Since the fusion protein is secreted into the bacterial medium, I pelleted the bacteria and mixed 10 ml of the supernatant with 10 ml Buffer A (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.3) and 425 ul 50% Ni Sepharose 6 Fast Flow suspension in a 50 ml tube. After 1 h of rotation at RT, I pelleted the resin and washed it twice with 10 ml Buffer A, re-suspended it in 0.5 ml Buffer A and transferred it to a 2 ml spin column. After spinning the buffer through with a 10 s spin in a mini-fuge (approx. 2,000 x g), I eluted the bound protein with 2 x 5 min incubations with 0.5 ml Buffer B (Buffer A with 500 mM imidazole), spinning the eluate into 2 separate tubes. To examine the efficiency of the purification process, I tested serial dilutions of the original bacterial sup, the sup after incubation with Ni Sepharose 6 Fast Flow (i.e. the non-binding proteins) and the 2 eluates in an ELISA against immobilized cyclophilin A, to which the peptide binds. I also ran the fractions on a 4-20% SDS gel and stained it with silver. As shown in the ELISA figure below, the first elution (E1) contains the majority of the peptide-BAP fusion, with a small amount in E2. Importantly, essentially all of the fusion protein was removed from the medium by incubation with the resin. The gel corroborates the ELISA data.
Overall, I was very pleased with the performance of Ni Sepharose 6 Fast Flow. The His-tagged protein was purified to a very high degree using the recommended buffers and a protocol that couldn’t be much simpler. I also purified the same fusion protein from the lysed bacterial pellet with the same results. The high protein binding capacity, fast flow rate, and super clean results, even with a simplified spin-column format, should make Ni Sepharose 6 Fast Flow ideal for high-throughput applications. The company also claims that it is ideal for scale-up. The price for the 25 ml size is $230, which is comparable to other IMAC resins on the market.
Michael Campa, Ph.D.
Asst. Research Professor of Radiology
Duke University Medical Center