Afnity Tags for Protein Purication

Joseph J. Falke and John A. Corbin

University of Colorado, Boulder, Colorado, USA

Selective immobilization of proteins greatly facilitates their purication, as well as their biochemical and biophysical characterization. When genetically fused to a target protein, a protein afnity tag provides a powerful tool to selectively capture and immobilize that target, in some cases providing single-step purication. Afnity tags consist of proteins or peptides with distinct amino acid sequences that are capable of a reversible, high afnity binding interaction with a specic partner molecule. The binding partner is linked to a large macroscopic particle or surface that renders it immobile, and thus it is readily amenable to manipulation. The highly specic interaction between the afnity tag and its cognate partner serves as the basis for selective capture of the target protein.

common and endows the target protein with two or more unique molecular handles.

AN

SELECTION CRITERIA AFFINITY TAG

FOR

Construction of a fusion protein begins with the selection of an afnity tag that is appropriate for the protein of interest (see below). The selection criteria include the size of the afnity tag, the organism that will be used to express the fusion protein, conditions for the immobilization or purication of the fusion protein, and the inuence of the afnity tag on the structural and functional characterization of the target protein.

Applications of Afnity Tags

Afnity tags have proven to be tremendously effective tools for a wide variety of applications, and they are now incorporated as a standard feature to reduce the number of steps in purication protocols developed for recombinant proteins. In addition, afnity tags have become indispensable in the immobilization of proteins for display on a surface, where the tag ensures that the fusion protein of interest is oriented with its functional regions exposed to docking with other macromolecules. Afnity tags are also used to detect and quantitate target proteins and to analyze protein protein or protein ligand interactions. Related technologies are developing rapidly, including bioreactors for multistep enzymatic reactions and bioadsorbants for extraction or degradation of toxic contaminants.

AFFINITY -FUSION GENE CONSTRUCTS

To incorporate the selected afnity tag, a suitable expression plasmid vector is chosen and recombinant DNA techniques are used to insert the target gene next to the afnity tag gene. Numerous vectors are commercially available that code for a selected afnity tag and a linker peptide, the latter often containing an imbedded protease recognition sequence. Additionally, an inducible promoter is present to enhance and regulate fusion protein expression, and a multiple cloning site region is included to facilitate insertion of the target gene with the fusion site at the C- or N-terminus as desired. In some cases, the coding region for a signal peptide is included to promote the secretion of the fusion protein into a specic cellular compartment or into the extracellular medium. Newly developed vectors fuse two different tags to each terminus of a target protein, thereby allowing two-step isolation schemes with enhanced purity and ensuring that only fully intact forms of the fusion protein are isolated.

Construction of a Fusion Protein

An afnity-tagged fusion protein typically consists of a single polypeptide chain with one or more afnity tags coupled to the C- or N-terminus of the target protein or inserted into a loop region. The coupling is via a peptide linker, usually up to 15 amino acids in length. If desired, the linker can contain a specic protease site for afnity tag removal. The use of multiple tags is increasingly

LINKER PEPTIDE COMPOSITION

The composition of the linker peptide can have a substantial impact on the function and utility of the fusion protein; therefore, a number of factors are considered during linker design. Typically, the linker is at least 5 10 residues long to allow free tumbling of

Encyclopedia of Biological Chemistry, Volume 1. q 2004, Elsevier Inc. All Rights Reserved.

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AFFINITY TAGS FOR PROTEIN PURIFICATION

the fusion and target proteins relative to one another, thereby preventing unwanted steric hindrance to binding events. For linkers containing a proteolytic cleavage site (typically for enterokinase, factor Xa, thrombin, or tobacco etch virus (TEV)), inclusion of 5 10 anking amino acids at both ends of the site ensures adequate accessibility to protease. Finally, the linker amino acid composition must be selected to minimize susceptibility to host organism proteases.

Overview of Fusion Protein Production and Immobilization

Purication of a recombinant fusion protein begins with the introduction of the expression plasmid into the host cell, followed by cell growth and induction of the fusion promoter. The host cells are lysed and the resulting lysate, containing the afnity-tagged protein, is incubated with a solid phase support to which the binding partner is coupled. Following binding of the tag to its immobilized partner, the support is washed to remove unbound components, yielding specic isolation of the tagged protein. If desired, the tagged protein can be released by either disrupting its interaction with the binding partner or by proteolysis of the linker region. Isolation and immobilization procedures often need to be optimized for each new afnity-tagged protein and expression host.

Afnity tag size can also impact target protein function and structural integrity. Small tags generally have less impact on protein structure and function and often need not be removed, while large tags can be perturbing and thus are typically removed prior to structural and functional studies. On the other hand, certain large tags can signicantly enhance the solubility of a fusion protein expressed at high levels; therefore, they are appropriate choices for the isolation of poorly soluble target proteins. Finally, when a target protein is toxic to the expression host, afnity tags that promote the aggregation of fusion protein into insoluble aggregates known as inclusion bodies are used. Key features of representative afnity tags are discussed below, and Table I presents a more comprehensive list.

PROTEIN LIGAND INTERACTION AFFINITY TAGS

Enzymes and small molecule-binding proteins are designed to bind their ligands with high specicity, thus many highly effective afnity tag systems are based on ligand-binding interactions. One of these is the widely utilized glutathione S-transferase (GST) tag (26 kDa) that binds with high specicity and low afnity (KD , 180 mM) to its substrate glutathione. Although the afnity of the GST tag for its ligand is low, commercially available immobilized glutathione matrices provide high local ligand concentrations that ensure adequate retention of the fusion protein. GSTtagged proteins are typically isolated from crude cell lysates by their interaction with immobilized glutathione on beads then eluted by competitive displacement with free, reduced glutathione. The low ligand afnity enables elution under mild, nondenaturing conditions. The GST tag often increases fusion protein solubility and stability, and it is ideally suited for overexpression of recombinant proteins in their native state. Often, the GST tag is proteolytically removed at the end of the purication due to its large size and tendency to form dimers. In other applications, the GST tag is retained and used to couple the target protein to a bead or surface for biochemical or biophysical studies. Another family of afnity tags that relies on protein ligand interactions are the cellulose-binding domains (CelBD). CelBDs are small, highly stable protein modules consisting of between 33 and 180 amino acids (3 20 kDa) that bind to different forms of cellulose or chitin with a broad range of afnities (KD , 0.01 to 400 mM). CelBD fusions can be constructed with afnity tags inserted internally or linked to either the C- or N-terminus of the protein of interest. Over 180 different CelBDs have been identied, providing a spectrum of polysaccharide specicities, binding afnities, and targeting properties. Certain CelBDs bind

Features and Types of Afnity Tag Systems

The large number of afnity tags currently available offers a diverse spectrum of biochemical properties that can be exploited for protein immobilization in a variety of contexts. Afnity tags range in size from short peptides less than 1 kDa to proteins as large as 120 kDa, and they can be classied into general categories based on their binding partner interaction such as protein ligand, polyamino acid matrix, antigen antibody, and protein protein. New types of tags based on novel binding partner interactions continue to emerge at a rapid pace. When choosing an afnity tag for a specic application, several features are considered. A subset of afnity tags possess the useful property of binding to their partner even under denaturing conditions. The ability to immobilize a tagged protein under denaturing conditions is a great advantage when the fusion protein is expressed in an insoluble or non-native form. Other tags utilize a binding partner interaction that is easily disrupted under mild conditions, thereby facilitating the recovery of target protein with full biological activity.

AFFINITY TAGS FOR PROTEIN PURIFICATION

59

cellulose essentially irreversibly, making them ideal for protein immobilization, while other CelBDs exhibit easily reversible binding. Denaturing conditions or proteolysis are often needed to elute irreversible CelBDs, while reversible CelBDs can be eluted under mild conditions such as the use of a competitive ligand (cellbiose or ethylene glycol) or desorption with water. Finally, CelBDs can be selected that target the fusion protein for secretion or inclusion body formation. The 51 amino acid chitin-binding domain (ChiBD) is derived from Bacillus circulans chitinase and exhibits high afnity, essentially irreversible binding to its ligand chitin, a natural carbohydrate polymer. ChiBD fusion proteins are immobilized under physiological conditions by adsorption to chitin coupled to a solid phase material. ChiBD fusion proteins often have an intein element incorporated into the linker region in place of a proteolytic recognition sequence. When remobilization of the fusion protein is desired, the intein element is activated and undergoes self-cleavage, resulting in the release of the target protein, while the intein-ChiBD region is retained on the solid phase. Similar inteins will probably soon be incorporated into the linkers of other afnity tags. Several other protein ligand afnity tag systems are currently in use, each having unique advantages. For example, the 40 kDa maltose binding protein (MalBP) is a large tag that often increases fusion protein solubility. MalBP binds to cross-linked amylose with moderate afnity (KD , micromolar range), permitting MalBP tagged fusion proteins to be immobilized in a reversible fashion. The full length MalBP tag directs a fusion protein to the oxidizing E. coli periplasm, where MalBP is a native protein and where the fusion protein may benet from disulde bond formation. Alternatively, removal of the MalBP leader peptide yields retention of the fusion protein in the cytosol, where the greater volume can allow higher expression levels.

6 histidine residues interspersed within a 19-residue polypeptide (KDHLI HNVHK EEHAH AHNK) that binds to specically Co2-carboxymethylaspartate and has a lower net charge than polyhistidine tags. The most widely employed metal chelator is iminodiacetic (IDA), while nitrilotriacetic acid (NTA) and carboxymethylated aspartic acid (trade name TALON) are also popular. The chelator is generally covalently coupled to polymer beads, or ferromagnetic beads for magnetic isolation. Adsorption of IMAC tagged proteins is normally performed at neutral to slightly basic pH to ensure that the histidine imidazole groups are not protonated. Mild elution conditions include ligand exchange with imidazole, extraction of the metal ion by a strong chelator like EDTA, or proteolytic elution. Low pH will also elute, but it can sometimes denature the target protein. IMAC is not recommended for target proteins possessing a metal center, since the metal can be stripped by the binding partner chelators. The Arg-tag is another example of a small polyamino acid afnity tag, in this case designed to raise the isoelectric point of the fusion protein to enhance its binding to a cation exchange matrix. Mild elution conditions are generally a NaCl gradient at alkaline pH. This tag also binds to at mica sheets, which may enable a variety of new applications.

ANTIBODY EPITOPE INTERACTION AFFINITY TAGS

Recombinant DNA technology has been employed to create peptide epitopes that bind well-characterized antibodies. This process is known as epitope tagging and is complementary to a more traditional approach where a novel antibody is generated for an existing epitope. Both approaches have been exploited to develop afnity tag systems based on the binding interaction between a peptide epitope and its specic antibody partner. In some cases, several antibodies with different properties are available for a given epitope. Proteins fused to peptide epitope afnity tags can be captured by immunoafnity interactions via immobilized monoclonal antibodies. Typical epitope tags range from 6 to 30 residues in length, are highly charged, and have little effect on protein structure function. They can be fused at either the C- or N-terminus, or even inserted within the protein of interest. Epitope tag expression vectors are commercially available for mammalian, insect, yeast, and bacterial host cells and provide a variety of tags including c-myc, FLAG, HA, T7, V5, VSV-G, recA, Protein C, Protein A, and Protein Z. Some of these are optimized for immobilization while others are primarily used for protein detection. The importance of epitope tags is illustrated by their many applications including analysis of in vivo protein expression,

POLYAMINO ACID MATRIX INTERACTION AFFINITY TAGS

Immobilized-metal afnity chromatography (IMAC) is a common technique used to purify recombinant proteins fused to a short peptide afnity tag. IMAC, which can be carried out under denaturing or nondenaturing conditions, relies on the interaction between multiple electron donors on the afnity tag with a transition metal ion (Co2, Ni2, Cu2, Zn2) chelated to a solid phase support. The afnity tag is typically polyhistidine, ranging 6 to 12 residues in length fused to the N- or C-terminus of the target, where 6-His is most common and the electron donor is the histidine imidazole ring. Recently, an afnity tag based on a natural peptide derived from the N-terminus of chicken lactate dehydrogenase has also been utilized. This HAT-tag contains

TABLE I Commonly Used Afnity Tags and Their Features

Nondenaturing Number of residues in tag 211 27189 Size of tag (kDa) 26 3 20 Afnity tag elution conditions (eluent) Yes (10 mM Reduced Glutathione) Yes (water) or No (Guanidinium HCL) Yes (10 mM Maltose) No

Category of tag Protein ligand interactions

Name of tag Glutathione S-transferase Cellulose-binding domains Maltose-binding protein Chitin-binding domain

Location of tag N-Term, C-Term N-Term, C-Term, Internal N-Term, C-Term N-Term, C-Term N-Term, C-Term, Internal C-Term

Immobile phase binding partner Glutathione Cellulose and other cellulosics Cross-linked amylose Chitin

Immobilization conditions Yes Yes

Expression hosta B, Y, I, M B, Y, I, M

396 51

40 5.6

Yes Yes

B B

Polyamino acid matrix interactions

Polyhistidine

6 12

0.81.7

Metal chelate

Yes or No (Denaturants) Yes

Yes (Imidazole) or No (Low pH) Yes (NaCl gradient at alkaline pH)

B, Y, I, M, P

Polyarginine

5 15

0.82.4

Cationexchange resin IgG

B, P

Antibody epitope interactions

Hemagglutinin (HA) T7 Synthetic protein A (Z domain)

1.1

N-Term, C-Term N-Term N-Term

Yes

No (Low pH)

Y, M

11 58

1.1 7

IgG IgG

Yes Yes

No (Low pH) No (Low pH)

B, Y, M, P, I B, Y, I, M, P

FLAG c-myc

8 11

1.0 1.2

N-Term, C-Term N-Term, C-Term N-Term, C-Term N-Term, C-Term N-Term, C-Term, Internal C-Term N-Term, C-Term N-Term, C-Term N-Term, C-Term

mAb M1 and mAb M2 IgG

Yes Yes

Yes (EDTA) Yes (free c-myc epitope) Yes (20 mM Biotin) Yes ( EGTA)

B, Y, I, M, P B, Y, I, M, P

Protein protein interactions

Biotin Calmodulinbinding peptide Strep-tag II

,20100 26

,2 11 3

Modied avidin or streptavidin Calmodulin

Yes Yes ( Ca2)

B, I, M B

1.1

Strep-tactin

Yes

Yes (desthiobiotin)

B, Y, I, M, P

Streptavidinbinding peptide Thioredoxin His-patch thioredoxin S-tag

38 109 109 15

4 11.7 11.7 1.8

Streptavidin Phenylarsine oxide-agarose Metal chelate S-protein

Yes Yes Yes or no Yes

Yes (biotin) Yes (b-mercaptoethanol) Yes (Imidazole) or No (Low pH) Yes (2 M Sodium Thiocyanate)

B B B B, I, M

B bacteria; Y yeast; I insect; M mammalian; P plant.

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AFFINITY TAGS FOR PROTEIN PURIFICATION

subcellular localization of gene products, determination of protein topology, cellular trafcking studies, and the investigation of protein protein interactions. They are also important in protein purication and immobilization. Antibody-binding partners are covalently coupled to agarose chromatography resins or magnetic glass beads. Immobilized antibodies tend to be less stable than many other afnity-binding partners due to their need for native structure. Elution under mild native conditions can be achieved by competitive displacement with free epitope, by proteolysis, or by exposure of the Ca2 dependent FLAG-tag to EDTA.

PROTEIN PROTEIN INTERACTION AFFINITY TAGS

Several high-afnity and high-specicity protein protein interactions identied in biological systems have been adapted for use as afnity tags. In vivo biotinylation enzymatically couples biotin, a small molecule vitamin, to a protein acceptor domain. The resulting modied protein provides an interesting example of a natural afnity tag. A number of well-characterized protein sequences are enzymatically biotinylated in vivo. These natural peptides of approximately 100 residues have been used as fusion tags that direct in vivo biotinylation of a fusion protein in a site-specic manner. Additionally, shorter synthetic peptides (approximately 20 residues) known as biotin acceptor peptides (BAPs) have been developed for use as biotin afnity tags. The high-afnity, specic interaction between biotin and either avidin or streptavidin (KD , 10215 M) serves as the basis for immobilization. Biotin-tagged fusion proteins are typically captured by binding to monomeric avidin or streptavidin coupled to a chromatography matrix or other surface. Elution with free biotin requires harsh conditions that can be detrimental to the target protein, but proteolytic elution can be carried out under mild conditions. Alternatively, the biotin-tag fusion can be immobilized to modied forms of avidin or streptavidin that exhibit lower biotin afnity. Elution can then be conducted under nondenaturing conditions by competitive displacement with free biotin or alkaline pH. Other examples of protein protein afnity tags include the Strep-tag, the calmodulin-binding peptide (CalBP), and the S-tag. The Strep-tag is a nine amino acid peptide that was developed as an articial ligand for streptavidin. Further renements to these molecules yielded the eight amino acid peptide Strep-tag II and a mutant form of streptavidin called Strep-Tactin capable of moderate-afnity binding (KD , 1 mM). Strep-tag fusion proteins are bound to streptavidin under physiological conditions and are efciently displaced by free biotin or a biotin derivative. The mild conditions

used for binding and elution enable Strep-fusions to be utilized in their native state. This small, stable, and nonperturbing tag is ideal for many applications in which a small tag is required, and for applications where detection of the fusion protein on a Western blot or by ELISA is desired. Moreover, since the Strep-tag is not itself a metalloprotein and its elution does not require metal chelators, it is well suited for metalloprotein applications. The 26 amino acid calmodulin-binding peptide (CalBP) was derived from the C-terminus of skeletal muscle light-chain kinase. The CalBP binds to calmodulin with high afnity (KD , 1 nM) in the presence of low CaCl2 concentrations ($ 0.2 mM). The binding partner, calmodulin, is commercially available linked to chromatography resins that are used to immobilize CalBP fusions. CalBP fusion proteins are eluted under mild conditions by a Ca2 chelating agent such as EGTA. CalBP fusions are primarily expressed in E. coli that lacks calmodulin family members and thus possesses no sequences evolved to bind to calmodulin. Eukaryotic cells are not recommended for expression of CalBP-tagged proteins due to their large number of endogenous proteins (approximately 30) that bind to calmodulin. The S-tag protein fusion system consists of the S-peptide and its binding partner the S-protein, both derived from RNAase A. The S-tag binds to the S-protein with moderate afnity (KD , 100 nM), resulting in a strong interaction that is inuenced by pH, temperature, and ionic strength. A unique feature of the S-tag system is that ribonucleolytic activity is restored when the S-peptide is bound to the 103 amino acid S-protein. This enzymatic activity is exploited to measure the molar concentration of the S-tag fusion protein down to 20 fM in a highly sensitive and rapid assay. S-protein-based reagents have been developed to probe SDS-PAGE blots for S-tag fusions employing colorimetric or chemiluminescent detection of the target protein down to nanogram quantities.

SEE ALSO

THE

FOLLOWING ARTICLES

Afnity Chromatography Two-Hybrid Protein Protein Interactions

GLOSSARY
epitope tag Short polypeptide fused to a protein of interest so that it will be recognized as a high-afnity binding target by a specic, well-characterized antibody. fusion protein Polypeptide made from a recombinant gene consisting of two or more gene fragments fused together. immobilized metal-afnity chromatography (IMAC) A type of afnity chromatography based on the specic interaction between a metal chelate stationary phase and a metal-binding peptide fused to a protein of interest.

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FURTHER READING
Bornhorst, J., and Falke, J. (2000). Purication of proteins using polyhistidine afnity tags. Methods Enzymol. 326, 245 254. Jarvic, J., and Telmer, C. (1998). Epitope tagging. Annu. Rev. Genet. 32, 601618. Nilsson, J., Stahl, S., Lundeberg, J., Uhlen, M., and Per-Ake, N. (1997). Afnity fusion strategies for detection, purication, and immobilization of recombinant proteins. Protein Expr. Purif. 11, 1 16. Sheibani, N. (1999). Prokaryotic gene fusion expression systems and their use in structural and functional studies of proteins. Prep. Biochem. Biotechnol. 29, 7790. Terpe, K. (2003). Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60, 523533.

BIOGRAPHY
Joseph J. Falke is Professor of Chemistry and Biochemistry, and Chair of the Molecular Biophysics Program, at the University of Colorado, Boulder. His research interests are in the area of signal transduction, in particular the mechanisms of cellular chemotaxis pathways in bacterial and eukaryotic systems. He holds a Ph.D. in chemistry from the California Institute of Technology and carried out his postdoctoral research at the University of California, Berkeley. His laboratory has made fundamental discoveries regarding the molecular mechanisms of receptors and signaling proteins involved in chemical sensing. John A. Corbin is a Postdoctoral Fellow in the Falke Laboratory at the University of Colorado, Boulder. He holds a Ph.D. in biology from the University of California, Santa Cruz. His research interests are in the general areas of protein structure, function, and mechanism.