Affinity chromatography theoretically does just that—a ligand that specifically interacts with the target protein is immobilized on a chromatography matrix; the target protein binds to the column, and unwanted proteins are eluted. In some cases, the affinity ligand is an antibody against the protein of interest; in others, the target protein is expressed from a plasmid that encodes for an "affinity tag" specific to a particular ligand.
This article discusses some of the affinity fusion systems available for recombinant proteins expressed in E. coli. A list of companies offering complete affinity fusion protein-purification systems (vectors and resins) is shown in the accompanying table.
Although the GST tag is more difficult to use than its popular competitor, the polyhistidine tag, many protein chemists prefer it because its use has been so well documented. "The six-His tag has become very popular, and a lot of scientists choose it, but ... a lot of people are still using GST because they are used to it," explains Krishna Mallia, a scientist at Rockford, Ill.-based Pierce Chemical Co. (www.piercenet.com). Companies offering GST purification systems and products include Pierce, Piscataway, N.J.-based Amersham Biosciences, and Madison, Wis.-based Novagen.
In recent years usage of polyhistidine fusion tags has eclipsed that of GST tags. First described by Jerker Porath and colleagues in the mid-1970s, this technique, called IMAC (immobilized metal ion affinity chromatography), exploits the affinity of histidine's imidazole side chains for metal ions such as nickel, zinc, and cobalt.2,3 IMAC generally uses tags composed of six histidine residues (6xHis) and a nickel-based affinity resin. His-tagged proteins adsorb to the column under neutral to alkaline pH conditions and are eluted at low pH or by competitive adsorption with imidazole.
Pierce's Mallia explains that the 6xHis tag has grown in popularity over the GST fusion tag, primarily because of its small size, which means the tag does not usually need to be cleaved from the fusion protein. Also, unlike GST-based purification systems, researchers can purify 6xHis fusions under denaturing conditions. Finally, the 6xHis tag is relatively non-immunogenic, a benefit when scientists inject the purified proteins into live animals.
Several companies sell complete IMAC systems. Valencia, Calif.-based QIAGEN's QIAexpress system uses an Ni2+-nitrilotriacetic acid (Ni-NTA) resin and offers vectors to fuse the tag to either the recombinant protein's N- or C-terminus. Novagen's His Bind® Purification Kits are similar, allowing purification of fusion proteins with six-to-10-histidine-residue-long tags. Novagen offers Ni-NTA- and IDA (iminodiacetic acid)-based resins on a variety of supports, including bulk agarose, magnetic agarose, and Fractogel®.
Carlsbad, Calif.-based Invitrogen's Xpress™ System is also a 6xHis-tag purification system. Its expression vectors, pTrcHis, encode for an enterokinase cleavage site to remove N-terminal tags. The company's ProBond™ metal affinity resin, included in the system, can be used for both gravity flow chromatography and FPLC. In contrast, QIAGEN's Ni-NTA resin comes in two different forms—one for gravity flow called Ni-NTA Agarose, and one with a more highly crosslinked support called Ni-NTA Superflow for FPLC.
Despite its many advantages, polyhistidine tags also present several pitfalls, says Yamuna Dasarathy, marketing manager at Amersham Biosciences, which offers affinity resins for purification of His-tagged proteins as well as fusion vectors and affinity resins for purification of GST-tagged proteins. These problems include the formation of inclusion bodies, difficulty in solubilization, lack of stability, and incorrect refolding of the fusion proteins. Further, nickel-based resins may be prone to leakage of metal ions, which can remain in the protein solution and damage the amino acid side chain of the target protein through oxidation. Finally, there is the potential for nonspecific binding on the IMAC column.
Palo Alto, Calif.-based BD Biosciences-CLONTECH's TALON™ system was designed to circumvent some of these problems. According to product manager Kate Marusina, the TALON affinity resin is a proprietary chelating ligand holding cobalt ions via four bonds, compared to Ni-NTA's three, producing tight ion binding and limited ion leakage. According to company literature, TALON is more specific to histidine-tagged proteins than nickel-based resins; as a result, fewer unwanted proteins are bound to the column after the sample is loaded. Marusina explains that the higher specificity is due to the three-dimensional structure of the cobalt-ligand complex; she compares it to a "large hand holding a large ion, which creates a spatial configuration that only allows binding of appropriately spaced histidine chains; that is, ones found in the affinity tag."
The TALON system is stable in up to 30 mM ß-mercaptoethanol and allows elution of proteins under mild, native, or denaturing conditions (pH 6-6.5 or 15 mM imidazole). In contrast, Ni-NTA resins require pH 4-5.5 or 150 mM imidazole for elution. CLONTECH offers pHAT™ vectors to generate polyhistidine fusion constructs. This vector's tag is a naturally occurring peptide sequence from chicken lactate dehydrogenase, which binds specifically to the TALON resin; it contains six histidine residues spaced unevenly between 12 other amino acid residues. Marusina explains that, unlike the synthetic 6xHis tag, the naturally occurring HAT tag is recognized as a native sequence and is thus less likely to form insoluble aggregates in inclusion bodies when expressed in E. coli.
While small size and low immunogenicity means that the His tag does not normally need to be removed after the target protein's purification, scientists performing structural studies or using purified product for therapeutics may still wish to do so. The TAGZyme™ system, recently introduced by QIAGEN, enables easy removal of N-terminal His tags. The system includes DAPase, which removes dipeptides sequentially from the N-terminus of purified His-tagged proteins until it reaches an engineered or intrinsic stop point (a glutamine residue introduced into the sequence serves as a DAPase stop point). To remove this additional glutamine residue, users add Qcyclase™, which catalyzes the formation of a pyroglutamate residue at the protein's N-terminus. This modified residue is removed by pGAPase™ treatment. Finally, the native protein can be isolated after an additional round of IMAC purification to remove the three enzymes; the entire process takes as little as 45 minutes. QIAGEN offers pQE-1 vectors that encode a glutamine residue between the histidine tag sequence and target protein sequence, and pQE-2 vectors for target proteins that contain an intrinsic DAPase stop point.
A Wealth of Options
La Jolla, Calif.-based Stratagene's Affinity system tags proteins with calmodulin-binding peptide (CBP). The tag is small (4 kDa), and users can elute the tagged proteins under mild conditions; CBP-tagged proteins bind to a calmodulin resin under low calcium concentrations and are eluted in neutral pH in the presence of 2 mM EGTA. Stratagene's pCAL vectors offer a variety of fusion options, including N- and C-terminal fusion, proteolytic cleavage of the tag, and inclusion of the FLAG epitope.
Another popular fusion method relies on the binding affinity between biotin and streptavidin or avidin, a system that prevents sample loss via oxidation from heavy metal ion usage. Proteins can be biotinylated by modification of lysines with biotin-ester reagents, but this technique can be difficult to use for affinity tagging, as more than one lysine residue may be modified.2 Site-specific biotinylation can be achieved by using a sequence that is naturally biotinylated in vivo, such as the C-terminal residues of the biotin carboxylcarrier protein (BCCP) of acetyl-CoA carboxylase.2 Madison, Wis.-based Promega's PinPoint vector encodes this tag; the resulting recombinant protein can be purified on a streptavidin-conjugated resin or with Promega's SoftLink™ Soft Release Avidin Resin. Whereas elution from avidin-conjugated resins often requires denaturing conditions because the interaction between biotin and streptavidin is so strong, SoftLink enables protein elution under mild conditions (5 mM biotin solution). Promega's PinPoint vectors also encode an endoproteinase Factor Xa proteolytic site to facilitate the fusion tag's removal.
Göttingen, Germany-based IBA GmbH's Strep-tag II™ expression and purification system, distributed in the U.S. by Sigma-Genosys of The Woodlands, Texas, also relies on the streptavidin-biotin interaction. The system employs pASK-IBA vectors that allow expression of N- or C-terminal fusion proteins that can then be purified by immobilization on a StrepTactin column. StrepTactin is a specially designed streptavidin derivative with a high binding affinity for Strep-tag II. The eight-residue Strep-tag II represents an improvement over its predecessor, the Strep-tag, which is nine amino acids long and is restricted to C-terminal fusions. Competitive elution of the fusion protein is achieved by adding small amounts of desthiobiotin, a biotin analog, to the washing buffer.
Beverly, Mass.-based New England BioLabs' pMAL™ System is yet another option, based on maltose-binding protein's (MBP) affinity for amylose. pMAL vectors include the malE gene, which encodes MBP, as well as a sequence coding for 10-residue spacer between MBP and the protein of interest. Additionally, pMAL vectors include protease-cleavage sites. One advantage of this system is that the MBP tag can improve protein solubility. Additionally, the malE signal sequence, included in the pMAL-2 vector series, directs the fusion protein through the cytoplasmic membrane to the periplasm, allowing recovery of the expressed protein from the periplasmic space. This can be advantageous in recombinant protein purification, as osmotic shock treatments can enrich for periplasmic proteins.3
New England Biolabs also offers the IMPACT™ (Intein-Mediated Purification with an Affinity Chitin-binding Tag) System, a novel affinity purification system that permits cleavage of the fused tag without a protease. The IMPACT-CN system allows C- or N-terminal fusion to the target protein. The system is based on the use of inteins, protein-splicing elements that undergo self-catalyzed cleavage from a larger protein. In vivo, the resulting spliced ends undergo subsequent fusion. This attribute has been modified in the IMPACT system so that site-specific cleavage occurs without subsequent ligation. IMPACT employs a dual tag composed of a modified intein, which can be induced to undergo self-cleavage under low temperature conditions in the presence of thiols or free cysteine, and a chitin-binding domain, which facilitates affinity purification via a chitin column.
NEB's IMPACT-TWIN (TWo-INtein) system fuses the target protein between two different intein tags and includes a chitin-binding domain on one or both of the intein tags. This multifunctional system allows use of single inteins for protein purification as in the IMPACT-CN system or generation of activated units for cyclization or polymerization. Cleavage of the tags can generate an N-terminal cysteine residue and a C-terminal thioester on the target protein, which can then undergo spontaneous condensation, creating a cyclic protein. This technique has been used to generate cyclic proteins up to 395 amino acids long.5
Novagen sells a number of specialized vectors encoding different affinity tags, in addition to its GST- and polyhistadine fusion products. Novagen's CBD•Tag™ system, which utilizes the company's pET CBD vectors, permits purification of proteins with a cellulose-binding domain tag. CBD fusion proteins can be purified using cellulose resins, such as Novagen's CBinD resins, and allow gentle elution by addition of ethylene glycol or under low salt conditions. Novagen's T7•Tag® purification system, designed to purify fusion proteins tagged with the first 11 amino acid residues of the T7 gene 10 protein, involves the binding of the tagged protein to T7•Tag monoclonal antibody covalently coupled to agarose. Elution occurs at low pH (2.2). The S•Tag™ purification system uses a 15 amino acid, hydrophilic peptide tag with a specific affinity for S-protein, which is derived from ribonuclease A; fused proteins can be purified with an S-protein agarose resin. The S•Tag rEK Purification Kit and S•Tag Thrombin Purification Kits enable cleavage of fusion tags from proteins containing an enterokinase cleavage site or a thrombin cleavage site, respectively. The S•Tag also offers a built-in detection system, as the association between the tag and the S-protein forms active ribonuclease and can be detected via a ribonuclease activity assay, allowing for sensitive quantitative measurement of any fusion protein.
The Future Looks Bright
Given affinity chromatography's high specificity and the huge popularity of affinity tags, it's only a matter of time before a new, improved method hits the market.
1. A. Constans, "Protein purification I: Liquid chromatography," The Scientist, 16:40, Jan. 21, 2002.
2. J. Nilsson et al., "Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins," Protein Expression and Purification, 11:11-16, 1997.
3. J. Porath et al., "Metal chelate affinity chromatography, a new approach to protein fractionation," Nature, 258:598-9, 1975.
4. T. Unger, "Show me the money: Prokaryotic expression vectors and purification systems," The Scientist, 11:20, Sept. 1, 1997.
5. T. Evans et al., "The cyclization and polymerization of bacterially expressed proteins using modified self-splicing inteins," Journal of Biological Chemistry, 274:18359-63, 1999.
6. K. Thorn et al., "A novel method of affinity-purifying proteins using a bis-arsenical fluorescein," Protein Science, 9:213-7, 2000.
7. B.A. Griffin et al., "Specific covalent labeling of recombinant protein molecules inside live cells," Science, 281:269-72, 1998.