The DNA sequence specifying a string of six to nine histidine residues is frequently used in vectors for production of recombinant proteins. The result is expression of a recombinant protein with a 6xHis or poly-His-tag fused to its N- or C-terminus.
Expressed His-tagged proteins can be purified and detected easily because the string of histidine residues binds to several types of immobilized metal ions, including nickel, cobalt and copper, under specific buffer conditions. In addition, anti-His-tag antibodies are commercially available for use in assay methods involving His-tagged proteins. In either case, the tag provides a means of specifically purifying or detecting the recombinant protein without a protein-specific antibody or probe.
Supports such as beaded agarose or magnetic particles can be derivatized with chelating groups to immobilize the desired metal ions, which then function as ligands for binding and purification of biomolecules of interest. This basis for affinity purification is known as immobilized metal affinity chromatography (IMAC). IMAC is a widely-used method for rapidly purifying polyhistidine affinity-tagged proteins, resulting in 100-fold enrichments in a single purification step.
The chelators most commonly used as ligands for IMAC are nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA). Once IDA-agarose or NTA-agarose resin is prepared, it can be "loaded" with the desired divalent metal (e.g., Ni, Co, Cu, and Fe). Using nickel as the example metal, the resulting affinity support is usually called Ni-chelate, Ni-IDA or Ni-NTA resin. The particular metal and chelation chemistry of a support determine its binding properties and suitability for specific applications of IMAC.
Affinity purification of His-tagged fusion proteins is the most common application for metal-chelate supports in protein biology research. Nickel or cobalt metals immobilized by NTA-chelation chemistry are the systems of choice for this application (see next section). In addition, different varieties of agarose resin provide supports that are ideal for His-tagged protein purification at very small scales (96-well filter plates) or large scales (series of chromatography cartridges in an FPLC system). When packed into suitable columns or cartridges, resins such as Ni-NTA Superflow Agarose provide for purification of 1 to 80 milligrams of His-tagged protein per milliliter of agarose beads. Compared to cobalt and other ligands used for IMAC, nickel provides greater capacity for His-tagged protein purification. Thermo Fisher Scientific offers HisPur Ni-NTA Superflow Agarose that exhibits a high dynamic binding capacity across a range of flow rates, making it an excellent choice for larger scale purifications.
High-yield, high-purity, medium-scale purification of 6xHisTagged protein. More than 4 grams of over-expressed 6xHis-GFP were purified in 3 hours using 200 mL columns containing HisPur Ni-NTA Superflow Agarose. One liter of lysate was loaded at a flow rate of 20 mL/min, then washed until baseline with wash buffer containing 30 mM imidazole. Bound protein was eluted with buffer containing 300 mM imidazole. Fractions containing purified 6xHis-GFP were pooled and quantitated using Pierce 660 nm Protein Assay (Cat. No. 22662). Load, flow-through, wash, and elute fractions were separated by SDS-PAGE, stained with Imperial Protein Stain (Cat. No. 24615) and evaluated using myImageAnalysis Software (Cat. No. 62237) to determine purity.
Poly-His tags bind best to IMAC resins in near-neutral buffer conditions (physiologic pH and ionic strength). A typical binding/wash buffer consists of Tris-buffer saline (TBS) pH 7.2, containing 10-25 mM imidazole. The low-concentration of imidazole helps to prevent nonspecific binding of endogenous proteins that have histidine clusters. (In fact, antibodies have such histidine-rich clusters and can be purified using a variation of IMAC chemistry.)
High concentrations of salt and certain denaturants (e.g., chaotropes such as 8 M urea) are compatible, so purification from samples in various starting buffers is possible. For this reason, it is best to use the His-tag for design and expression of recombinant proteins that may need to be purified in denatured form from inclusion bodies. (Contrast this with the GST-tag, which is an enzyme that must remain functional to enable purification.) It is important to note that EDTA and reducing agents such as DTT and TCEP can adversely affect the performance of regular Ni-IMAC supports by stripping off the metal. But a specially engineered Ni-IMAC chemistry is available that can tolerate the presence of reducing agents and chelators such as EDTA at higher concentrations without the loss of performance. The EDTA compatible Ni-IMAC chemistry is available in magnetic bead (Cat. No. A50588) and resin (Cat. No. A50585) formats. They are specifically suited for purifying expressed His-tagged proteins that are secreted into cell culture media, or for purifying intracellular His-tagged proteins that need the presence of EDTA to maintain stability and function.
Elution and recovery of captured His-tagged protein from an IMAC column is accomplished by using a high concentration of imidazole (at least 200 mM), low pH (e.g., 0.1 M glycine-HCl, pH 2.5) or an excess of strong chelators (e.g., EDTA). Imidazole is the most common elution agent.
Be aware that immunoglobulins are known to have multiple histidines in their Fc region and can bind to IMAC supports. High background and false positives can result if binding conditions are not sufficiently stringent (i.e., with imidazole) and the immunoglobulins are abundant relative to the His-tagged proteins of interest. Albumins, such as bovine serum albumin (BSA), also have multiple histidines and can bind to IMAC supports in the absence of His-tagged proteins in the sample or imidazole in the binding/wash buffer.
Thermo Scientific HisPur Cobalt Resin is a tetradentate chelating agarose resin charged with divalent cobalt (Co2+). The resin provides a high degree of purity and may recover more than 10 mg of pure His-tagged protein per milliliter of resin without metal contamination or the need to optimize imidazole washing conditions. .
Affinity purification of His-tagged proteins. Cell lysate containing over-expressed recombinant 6xHis-tagged Green Fluorescent Protein (GFP) was prepared in B-PER Bacterial Protein Extraction Reagent (Cat. No. 78243) and protease inhibitors. Protein concentrations were determined by Coomassie Plus Protein Assay (Cat. No. 23238). Bacterial lysate (1.0 mg total protein) was applied to a 0.2 mL bed volume of HisPur Cobalt Resin in a spin column. The resin was washed three times with 0.4 mL of wash buffer containing 10 mM imidazole. His-tagged proteins were eluted three times with 0.2 mL of elution buffer containing 150 mM imidazole. Gel lanes were normalized to equivalent volume. Gel was stained with Imperial Protein Stain (Cat. No. 24615). M = Molecular Weight Marker, L = lysate load, FT = flow-through.
This 32-page handbook provides useful information on our broad portfolio of reagents and tools for protein extraction, clean-up, immunoprecipitation and purification. Practical information, selection guides, and relevant data are included to help you improve your protein yield and downstream analysis.
Specific topics covered include the following:
Nickel is the most widely available metal ion for purifying His-tagged proteins. Nickel generally provides good binding efficiency to His-tagged proteins but also tends to bind nonspecifically to endogenous proteins that contain histidine clusters. As stated above, a small amount of imidazole in the binding/wash buffer helps to control off-target binding.
Cobalt exhibits a more specific interaction with histidine tags, resulting in less nonspecific interaction. For this reason, cobalt is the preferred divalent cation for purifying His-tagged proteins when high purity is a primary concern. Thermo Fisher Scientific offers both nickel and cobalt IMAC resin formats. See below.
Ni-NTA and Cobalt resins maximize yield and purity, respectively. Gel panels: Bacterial lysate containing over-expressed 6xHis-AIF2 (6 mg total protein) or 6xHis-GFP (4 mg total protein) was applied to HisPur Ni-NTA Resin (0.2 mL) and purified by the batch-bind method. The same amount of total protein was applied to Ni-IDA and HisPur Cobalt resins and purified according to the manufacturer’s instructions. All Gels lanes were normalized to equivalent volume. M = molecular-weight marker, L = lysate load and FT = flow-through.
Copper ions bind his-tags more strongly than cobalt or nickel. This provides the highest possible binding capacity but also the poorest specificity. For this reason, copper IMAC is commonly used only for binding applications in which purification is not the objective (e.g., plate-coating of an already-purified His-tagged protein for use in an assay).
Besides affinity purification, other applications for His-tagged fusion proteins are made possible with the aid of IMAC-type chemistries or His-tag-specific antibodies:
Superior performance of Pierce High-Capacity Ni-IMAC MagBeads, EDTA Compatible compared to magnetic beads from supplier C when purifying proteins from cell culture supernatant. Cell culture supernatant (Expi293) containing over-expressed His-tagged EPO (40 mg total protein) was applied to “Pierce High-Capacity Ni-IMAC MagBeads, EDTA Compatible” as well as Competitor C. Beads were processed using protocols with buffers recommended by the manufacturers. Binding was performed with all samples for 30 minutes. The beads were collected on a magnetic stand and the flow-through (unbound) fractions were saved for analysis. The beads were then washed twice, and bound protein was eluted with a 15-minute incubation in Elution Buffer. The eluates were resolved on an SDS-PAGE gel and stained with GelCode Blue (Cat. No. 24594). Gel lanes were normalized by volume. M = MW marker, L = lysate load, FT = flow-through, W = wash, and E = elution.
*HC = High-capacity
Efficient, high-purity yield of His-tagged protein expressed into Expi293 cell culture media. Cell culture supernatant containing over-expressed His-tagged HSA (50 mg total protein) was applied to Pierce High-Capacity Ni-IMAC resin, EDTA Compatible (1 mL column) and purified by FPLC. Sample was loaded at a flow rate of 0.5 ml/min, then the column was washed until baseline with wash buffer containing 20 mM imidazole. Bound protein was eluted with buffer containing 500 mM imidazole. The eluates were resolved on an SDS-PAGE gel and stained with GelCode Blue (Cat. No. 24594). M = MW marker, L = lysate load, FT = flow-through, W = wash, and E = elution.
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