REVIEW

The tandem affinity purification technology: an overview

Yifeng Li

Received: 21 February 2011 / Accepted: 8 March 2011

� Springer Science+Business Media B.V. 2011

Abstract Tandem affinity purification (TAP) is a

methodology for the isolation of protein complexes

from endogenous sources. It involves incorporation

of a dual-affinity tag into the protein of interest and

introduction of the construct into desired cell lines or

organisms. Using the two affinity handles, the protein

complex assembled under physiological conditions,

which contains the tagged target protein and its

interacting partners, can be isolated by a sequential

purification scheme. Compared with single-step puri-

fication, TAP greatly reduces non-specific background

and isolates protein complexes with higher purity.

TAP-based protein retrieval plus mass spectrometry-

based analysis has become a standard approach for

identification and characterization of multi-protein

complexes. The present article gives an overview of the

TAP method, with a focus on its key feature—the dual-

affinity tag. In addition, the application of this

technology in various systems is briefly discussed.

Keywords Affinity tag � High-recovery � Protein

complex � Tandem affinity purification

Introduction

Tandem affinity purification (TAP), developed by

Rigaut et al. (1999), is a generic approach for the

purification of protein complexes. It was initially

tested in yeast and soon found to be applicable to

other cells or organisms. The key strategy involves

fusion of two affinity modules (the TAP tag) to the

protein of interest and introduction of the construct

into the host cell or organism. The dual-affinity tag

allows the expressed target protein along with its

interacting partners to be purified from cell extracts in

two consecutive steps. Compared with single-step

purification, TAP significantly reduces non-specific

background. This method has proved superior to the

yeast two-hybrid approach because it is more sensi-

tive, less error-prone and capable of disclosing multi-

component interactions (Rigaut et al. 1999; Puig et al.

2001; Gavin et al. 2002). In combination with mass

spectrometry for protein identification, TAP technol-

ogy constitutes a powerful tool for the characteriza-

tion of protein complex associated with a given target

(Bauer and Kuster 2003).

Rigaut et al. (1999) chose two IgG-binding units of

protein A of Staphylococcus aureus (ProtA) and the

calmodulin-binding peptide (CBP) to construct the

TAP tag after testing several commonly used affinity

tags that also include the FLAG tag, the Strep tag, the

His tag, and the chitin-binding domain (CBD),

because only the two selected tags allowed efficient

recovery (roughly 80 and 50%, respectively) of a

Y. Li (&)

Protein Production Core Facility, Department of

Biochemistry, University of Texas Health Science Center

at San Antonio, Medical Building 431B,

7303 Floyd Curl Drive, San Antonio, TX 78229, USA

e-mail: liy10@uthscsa.edu

123

Biotechnol Lett

DOI 10.1007/s10529-011-0592-x

fusion protein present at low concentration. In

addition to ProtA and the CBP, the TAP tagging

cassette contains a tobacco etch virus (TEV) protease

cleavage site between the two affinity modules

(Fig. 1a). This TEV cleavage site is included to

allow proteolytic release of the IgG-bound material

under native conditions because otherwise ProtA

can only be released from IgG under denaturing

conditions at low pH. TEV protease is highly specific

and few cellular proteins contain its recognition

sequence; therefore cleavage of the target or its

associated proteins by this enzyme is expected to be

rare. Once expressed in cells, the TAP-tagged protein

forms complex with its endogenous partners. In

general, the target-containing protein complex is first

recovered on the IgG matrix (via the ProtA moiety).

After washing, the bound material is released by TEV

cleavage. The eluate is then incubated with calmod-

ulin resin in the presence of calcium. Finally, the

captured material is released by adding chelating

agents (e.g., EGTA) (Fig. 1b). The second purifica-

tion step removes contaminants remaining after the

first purification as well as the TEV protease. For

purification from yeast extracts, the two purification

steps can be performed in the reverse order, except

that in this case the final purified fraction will be

contaminated with the TEV protease (Puig et al.

2001). In mammalian cells, however, using the

calmodulin resin first is not recommended because

many endogenous proteins possess calmodulin-bind-

ing capacity and can be copurified. In yeast, the

ProtA-CBP tandem tag allows roughly 20–30% of the

target protein to be recovered, and in most cases

sufficient amount of complexes for protein identifi-

cation by mass spectrometry can be obtained from 2 l

of culture (Seraphin et al. 2002; Dziembowski and

Seraphin 2004).

In the original design, the TAP tag was fused to

the C-terminus of the target protein. Due to its

relatively large size (*21 kDa), in certain cases the

tag is found impair protein function, resulting in

altered phenotype or unviable strains (Puig et al.

2001; Gavin et al. 2002). This problem can some-

times be overcome by switching the tag to the

N-terminus (Puig et al. 2001). N- and C-terminal tags

may also have different effects on protein expression

level, and have different chances of being exposed.

Ideally, the terminus to which the tandem tag is

placed should be determined empirically. In either

N- or C-terminal tagging, the CBP is adjacent to the

protein of interest whereas the ProtA module is

located at the extreme terminus of the fusion (Puig

et al. 2001) (Fig. 1a). As a slightly varied version of

the original TAP method, the two affinity modules,

a

b

Fig. 1 The original TAP strategy. a Schematic representation

of C- and N-terminal fusion constructs. The classical TAP tag

is composed of ProtA and the CBP with a TEV protease

cleavage site in between. Due to the purification strategy, in

either construct ProtA is located at the protein extremity.

b Schematic overview of the TAP procedure. The TAP tagged

bait protein along with its interacting partners is sequentially

purified using IgG matrix and calmodulin resin. Release of

IgG- and calmodulin-bound protein complex is achieved by

using TEV protease and chelating agents, respectively

Biotechnol Lett

123

ProtA with TEV site and the CBP, can be separately

attached to two different proteins of the same

complex (Rigaut et al. 1999; Puig et al. 2001). This

split-tag strategy guarantees that the final purification

is enriched with protein complex containing both

components (Puig et al. 2001; Tharun 2008).

Diverse TAP tags

After having been proved successful in yeast, the TAP

method was quickly adapted to other cells or organ-

isms (Cox et al. 2002; Rivas et al. 2002; Forler et al.

2003; Gully et al. 2003). However, despite its

strength, the original TAP tag has limitations and

disadvantages. For instance, the protein recovery in

higher eukaryotes is usually much lower than that in

yeast (Drakas et al. 2005; Schimanski et al. 2005;

Burckstummer et al. 2006; Yang et al. 2006; Gunzl

and Schimanski 2009). In particular, the calmodulin

affinity step has been found to be inefficient (Schi-

manski et al. 2005; Schaffer et al. 2010). Endogenous

calmodulin and calmodulin-binding proteins in mam-

malian and insect cells may interfere with binding of

the target, causing poor protein recovery. However,

considering that most of the calmodulin-binding

proteins can be removed during the first purification

step, endogenous calmodulin might be the major

cause of inefficient binding to the calmodulin resin.

Accordingly, the efficiency of the calmodulin affinity

step likely depends on the relative amounts of free

endogenous calmodulin and the target protein (Schi-

manski et al. 2005); that means whereas the amount of

free calmodulin is probably not enough to fully block

the binding of a high-abundant protein, it may be

sufficient to completely block the binding of a low-

abundant protein. This is consistent with the observa-

tion that the calmodulin affinity step is efficient in

some cases but not in others. Nevertheless, in certain

cases the low recovery could be simply due to

inefficient elution from the calmodulin beads despite

good binding (Zeghouf et al. 2004). Another disad-

vantage associated with calmodulin affinity purifica-

tion is that the chelating agent used for elution can

irreversibly interfere with the function of cation-

dependent proteins. In addition to the disadvantages

regarding the use of the CBP, the relatively large size

of the original TAP tag is a drawback as it increases

the chance of impairing protein function.

As the TAP technique has been widely exploited,

more than 30 alternative tags, using different com-

binations of affinity handles, have been developed

(Li 2010). Some of them effectively overcome the

disadvantages of the original ProtA-CBP tag and

achieve improved protein recovery and/or flexibility.

Twelve of these alternative TAP tags, including two

commercial systems, eight high-recovery tags, two

unique tags that enable stringent washes and protein

localization, respectively, are briefly discussed in the

following sections (Fig. 2) (Table 1).

Commercial products

InterPlay Mammalian TAP system

The InterPlay Mammalian TAP system was developed

by Stratagene, to whom a patent has been granted

(Braman et al. 2007). The system is specially designed

to recover target- interacting proteins in mammalian

cells using the TAP method. The two affinity tags used

by this system are the streptavidin-binding peptide

(SBP) and CBP (Fig. 2a). The SBP is a 38-residue

peptide that binds to streptavidin with high affinity and

can be specifically eluted by biotin under mild

conditions. Stratagene provides pNTAP and pCTAP

expression vectors that allow the SBP-CBP tandem tag

to be fused to the 50- and 30-ends of the target gene,

respectively. The latest version of the system, Inter-

Play Adenoviral TAP system, also includes competent

cells carrying the pAdEasy-1 plasmid, a viral-based

gene delivery system that facilitates introducing of

the target gene to an increased number of cells. A

purification kit containing the corresponding affinity

resins and relevant buffers is separately available from

the same company.

The main advantage of the system is that the

fusion proteins can be eluted from both resins with

small molecules under mild conditions, and therefore

protease cleavage, a step may cause significant loss of

yield, is not required to release the captured protein

complexes. It is noteworthy that the order in which

the two purification steps are performed is critical.

For best results, the protein complex should be

purified by streptavidin resin first. This is because

mammalian cells contain many endogenous calmod-

ulin-binding proteins, which will be copurified with

the target if the calmodulin resin is used first. Various

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123

protein complexes have been successfully isolated

from mammalian cell lines using the InterPlay TAP

system (Chiu et al. 2006; Haag Breese et al. 2006;

Bradley et al. 2007; Gallois-Montbrun et al. 2007;

Griffin et al. 2007; Medina-Palazon et al. 2007; Wei

et al. 2007; Wiederschain et al. 2007; Conner and

Wang 2008; Ahlstrom and Yu 2009; Juillard et al.

2009; Xie et al. 2009; Hentschke et al. 2010; Sharma

et al. 2010; Wang et al. 2010; Park et al. 2011).

However, despite its wide use, the overall recovery of

this system is not provided by any of these studies.

FLAG HA system

FLAG and haemagglutinin (HA) are small epitope

tags. Fusion proteins containing them can be purified

by affinity resins derived from tag-specific antibod-

ies. Ogawa et al. (2002) first used a FLAG-HA

tandem tag to isolate protein complexes from HeLa

cells, and a detailed protocol is separately available

(Nakatani and Ogryzko 2003). Ye et al. (2004a;

2004b) shortly after used a slightly varied version,

FLAG-29 HA, to achieve the same goal. The authors

made both N- and C-terminally tagged constructs for

each target protein and found that the relative

efficiency of these two fusions in forming complex

with endogenous partners is target protein-dependent.

In 2006, Zenser et al. (2008) at Sigma-Aldrich

filed a patent application covering the TAP system

based on the FLAG-HA tag (Fig. 2b). Sigma–Aldrich

now provides commercial kits (FLAG HA TAP Tag

Generation Kit and FLAG HA Tandem Affinity

Purification Kit) that allow efficient generation and

isolation of FLAG-HA dual-tagged fusion proteins.

The small size and non-eukaryotic nature of the

FLAG-HA tag is an advantage, as these features

minimize the chance of interfering with complex

assembly or protein function. Similar to the InterPlay

system, no protease treatment is required for elution.

a

c

d

e

f

g

h

i

j

k

l

b

Fig. 2 Schematic representation of 12 alternative TAP-tag-

ging systems. Filled triangle indicates specific protease

cleavage sites. Except for the LAP tag, only one option

(N- or C-terminal fusion depending on the initial description or

relative popularity) is shown for all the other tags. However, in

all cases it is possible to attach the tag to the alternative end of

the bait protein. a The SBP-CBP tag used by the InterPlay

Mammalian TAP system. b The FLAG-HA tag. Commercial

kits for the generation and isolation of tagged proteins are

available from Sigma-Aldrich. c The 39 FLAG-His tag, which

is highly efficient for isolating protein complexes from

Drosophila cells and tissues. d–f Three TAP tags in which

the CBP moiety in the original TAP tag was replaced by ProtC,

SBP and 29 FLAG, respectively (ProtG is similar to ProtA).

All of them showed improved efficiency. For the same reason

as that given for the original tag, a TEV cleavage site is

included in all three constructs. g The His-29 Strep II tag

which contains two TEV cleavage sites (the second one is

added to improve cleavage efficiency). TEV cleavage was used

to release protein bound to the Ni-NTA resin. h The 29 Strep

II-FLAG. i The SBP-HA tag. j A modified version of

Stratagene’s SBP-CBP tag. The SBP and a newly added

C-terminal His tag are used for the first and second purification,

respectively. k The His-biotin tag that allows both purification

steps to be carried out under fully denaturing conditions. l The

LAP tag. Both N- and C-terminal tag use GFP for the initial

purification. However, the protease cleavage sites and the

second affinity epitopes are different in each case. In particular,

the N-terminal tag uses TEV site and S-peptide whereas the

C-terminal uses HRV3C site and His tag

b

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123

Captured proteins can be efficiently eluted from the

anti-FLAG and anti-HA affinity resins with 39 FLAG

and HA peptide, respectively. In addition to the

examples mentioned above, the FLAG-HA tandem

tag has been applied in several other cases, allowing

efficient isolation of sufficient amount of various

protein complexes from mammalian cells for mass

spectrometry analysis (Shi et al. 2003; Tagami et al.

2004; Di et al. 2008; Shim et al. 2008; Skaar et al. 2009;

Chen et al. 2010; Lewis et al. 2010, Nittis et al. 2010;

Takai et al. 2010; Wen and Damania 2010). Sigma’s

FLAG HA TAP Tag Generation Kit, however, only

allows generation of N-terminally tagged proteins. In

practice, however, the two affinity tags are sometimes

tandemly fused to the C-terminus (Tagami et al. 2004;

Lewis et al. 2010) or put separately at both ends of

the target protein (Di et al. 2008).

High-recovery tags

39 FLAG-His

Kaneko et al. (2004) first demonstrated the use of

FLAG-His tandem tag when purifying a septin protein

complex from C. albicans. In their approach, the

clarified cell lysates were first subjected to anti-FLAG

agarose and the eluate was subsequently applied to

Ni–NTA resin. Shortly after Yang et al. (2006)

switched to a 39 FLAG-His tag for protein complex

purification from Drosophila tissues when the original

ProtA-CBP tag failed to give satisfactory results

(Fig. 2c). Sequential purification was performed in the

same order as did by Kaneko et al. (2004). The authors

showed that the 39 FLAG-His tag is more effective

than ProtA-CBP for isolating protein complexes from

Drosophila cells and tissues. For three bait proteins,

the percentage recovery of 39 FLAG-His purification

was consistently in the 10-20% range, which was an

order of magnitude higher than that of ProtA-CBP.

The high recovery of the 39 FLAG-His tag enabled

identification of several putative cofactors of Dro-

sophila nuclear receptor family proteins by mass

spectrometry. This FLAG-His combination has also

been used to isolate protein complexes from mam-

malian cells (Saade et al. 2009).

ProtA-Protein C (ProtC)

When applying the TAP method to trypanosomes,

Schimanski et al. (2005) found that the procedure

Table 1 General features of the TAP tags discussed in the text

TAP tag Approximate size (kDa) Recovery (%) Cleavage site References

ProtA-CBP 21 20–30a TEV Rigaut et al. (1999)

SBP-CBP 8 NAb Nonec Braman et al. (2007)

FLAG-HA 3 NA None Zenser et al. (2008)

33 FLAG-His 3 10–20 None Yang et al. (2006)

ProtA-ProtC 19 10–20 TEV Schimanski et al. (2005)

ProtG-SBP 19 5 TEV Burckstummer et al. (2006)

23 FLAG-ProtA 19 5–30 TEV Tsai and Carstens (2006)

His-23 Strep II 6 16 23 TEV Giannone et al. (2007)

23 Strep II-FLAG 5 27–48 None Gloeckner et al. (2007)

SBP-HA 5 30–40 None Glatter et al. (2009)

SBP-His 8 ? 1d [50 None Li et al. (2011)

His-biotin 10 NA None Tagwerker et al. (2006)

GFP-S/Hise 36 NA TEV/HRV3Cf Cheeseman and Desai (2005)

a The average value in yeast (the corresponding value in higher eukaryotes is usually much lower)b NA not availablec Enzymatic cleavage is not requiredd The N-terminal SBP-CBP tag plus the C-terminal His tage The N- and C-terminal tags use S-peptide and His tag, respectively, as the second affinity handlef The N- and C-terminal tags use TEV and HRV3C protease sites, respectively, to cleave off the GFP moiety

Biotechnol Lett

123

based on the original ProtA-CBP tag did not yield

enough recovery for protein identification. In partic-

ular, they believed that the low yield was due to the

inefficiency of the calmodulin affinity purification

step and their conclusion was confirmed in personal

communication with other researchers in the field. To

overcome this problem, the authors replaced the CBP

with protein C epitope (ProtC), a 12-amino-acid

peptide derived from human protein C (Fig. 2d).

ProtC binds with high affinity to anti-ProtC antibody

HPC4 in the presence of calcium and elution can be

achieved with either chelating agents or ProtC

peptide.

Using TbSNAP50, a subunit of the small nuclear

RNA activating protein complex (SNAPc), as the bait

protein, TAP based on the ProtA-ProtC tag allowed

isolation of a functional transcription factor complex

from human parasites T. brucei (Schimanski et al.

2005). Both IgG and anti-ProtC matrixes are highly

efficient at capturing the tagged proteins ([90 and

[80%, respectively). TEV protease cleavage and

EGTA elution recovered 30–40 and 50% of the

protein bound to the corresponding matrix, respec-

tively. Overall, 10–20% of the target protein present

in the input material was recovered in the final eluate.

In addition, by doing a parallel study using the ProtA-

CBP tag the authors further confirmed that the low

efficiency of the original procedure was due to the

calmodulin affinity purification. The authors also

indicated that although the experiments were con-

ducted with trypanosomal extracts, the method does

not contain trypanosome-specific features except for

the extract preparation. Furthermore, protein C is

only expressed in hepatocytes and its HPC4-binding

site is not well conserved among mammals. There-

fore, specific detection and purification of ProtA-

ProtC tagged proteins by the HPC4 antibody should

be feasible in any non-hepatocyte cell lines. Indeed,

ProtA-ProtC tag has been used to obtain UL97

(a protein kinase encoded by human cytomegalovi-

rus) complexes from infected human foreskin fibro-

blasts (HFF) cells (Kamil and Coen 2007).

Protein G (ProtG)-SBP

After realizing that the low overall yield has made

the application of TAP in mammalian cells unsatis-

factory, Burckstummer et al. (2006) designed sev-

eral dual-affinity tags aimed at improving protein

recovery. One of these tags, which is based on ProtG

and the SBP (Fig. 2e), resulted in a ten-fold increase

in yield, allowing purification and identification of the

Ku70-Ku80 protein complex from 5 9 107 HEK293

cells. ProtG is an IgG-binding protein similar to ProtA

but shows a slightly higher affinity. For the same

reason as that given for the original tag, TEV-protease

cleavage is required to release IgG-bound protein

under native conditions. The authors estimated

that the first binding caught 40% of the bait and

about 30% of the captured protein can be retrieved

after TEV-protease cleavage. The second binding

recovered approx. 75% of the remaining material

and on average 50% of the streptavidin-bound target

can be found in the biotin eluate. Overall, the TAP

procedure using the ProtG-SBP tag recovers about

5% (40% 9 30% 9 75% 9 50% = 4.5%) of the bait

protein present in the lysate. Consistent with its

superior performance in mammalian cells, the ProtG-

SBP tag has been shown to outperform the original

TAP tag with respect to both yield and specificity in

insects and plants (Kyriakakis et al. 2008; Van Leene

et al. 2008).

The ten-fold increase in protein yield achieved by

the ProtG-SBP tag is a remarkable improvement.

However, the overall percentage recovery (i.e., 5%) is

still relatively low and there are likely three factors

that prevent further improvement. First, the initial

IgG binding is not quite efficient and only catches

40% of the bait. Second, the TEV-protease cleavage

step causes significant loss of the yield. Third, the

biotin elution is not complete and half of the captured

protein remains bound to the streptavidin resin.

Modification at these three points shall make higher

recovery possible. As will be shown in the following,

this is really the case.

29 FLAG-ProtA

The 29 FLAG-ProtA tag is another example in which

the CBP in the original TAP tag is substituted by

other affinity modules to improve protein recovery

(Tsai and Carstens 2006) (Fig. 2f). The reason that

the FLAG tag was chosen is that it gives good

recovery of the tagged proteins (i.e., 80%) based on

the authors’ experience. Like in the original TAP tag,

a TEV protease cleavage site is included to allow

proteolytic release of the IgG-bound material. For

both purification steps, binding and elution can be

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123

carried out under gentle, nondenaturing conditions. It

was estimated that the protein recovery on the IgG

and anti-FLAG resins was 50–80% and 80%, respec-

tively. The overall yield is expected to be in the range

of 5–30% when other losses via washes and TEV

cleavage are accounted. A similar tag which contains

a single copy of FLAG was previously developed by

Knuesel et al. (2003), and it allowed successful

purification of the SMAD3 protein complex from

mink lung cells. However, percentage recovery of the

target protein fused with this tag is not provided.

His-29 Strep II

Also in an effort to increase bait protein recovery,

Giannone et al. (2007) evaluated the efficacy of several

dual-tags (i.e., 33 HA-ProtA, His-23 Strep II and

ProtA-23 Strep II) using human telomeric repeat

binding factor 2 (TRF2) as the bait protein. They found

that all these tags yielded sufficient amount of protein

for mass spectrometry analysis, and recovered TRF2

and its known interacting partners. In particular,

the His-23 Strep II tag (Fig. 2g), which allows the

fusion protein to be sequentially purified using

Ni-NTA and Strep-Tactin resins, produced the best

TRF2 sequence coverage and identified the most

known TRF2 interacting proteins relative to others

tags. Rather than using imidazole, elution from Ni-

NTA resin was mediated by TEV cleavage. In fact, the

authors incorporated two instead of one TEV sites to

improve cleavage efficiency. In the case of TRF2,

approximately 16% of the target protein was recovered

in the final eluate, enabling identification of protein

complex components from as little as 7 9 107 cells.

29 Strep II-FLAG

The TAP tag consisting of a tandem Strep II and

FLAG was developed by Gloeckner et al. (2007)

(Fig. 2h), who concerned that the original 21-kDa

ProtA-CBP tag may have a risk of impairing protein

function and binding. A similar tag that contains a

single copy of Strep II was previously used to purify

protein complexes from bacteria (Fodor et al. 2004).

In both purification steps, proteins bound to the

corresponding affinity matrix (i.e., Strep-Tactin resin

and anti-FLAG agarose) can be efficiently released

via competitive elution under native conditions. A

step-by-step guide for generating and purifying 29

Strep II-FLAG tagged proteins from mammalian cells

can be found in two separate papers (Gloeckner et al.

2009a, b).

The efficiency of the TAP based on 29 Strep II-

FLAG was determined for three bait proteins, and the

estimated recovery rate ranges from 27 to 48%

(Gloeckner et al. 2007). The amount of cells used for

purification can be varied depending on the expres-

sion levels of the bait protein and usually 4 9 108

cells is a good starting point (Gloeckner et al. 2009b).

Using 29 Strep II-FLAG tagged lebercilin as a bait,

den Hollander et al. (2007) identified 24 proteins that

link the target protein to centrosomal and ciliary

functions.

SBP-HA

Unsatisfied with the protein yields from mammalian

cells given by existing TAP procedures, Glatter et al.

(2009) developed a double-affinity purification pro-

tocol based on a novel dual-affinity tag composed of

SBP and HA (Fig. 2i). The target-containing protein

complex was sequentially purified by Strep-Tactin

beads and anti-HA agarose. It was estimated that the

first purification step and the overall procedure

recovered [90 and 30–40% of the bait protein

present in the cell lysate, respectively. The authors

believed that efficient binding and elution in both

steps as well as the elimination of the TEV cleavage

step contributed to the high yield. For the particular

bait protein used in their study, which is an abundant

phosphatase, complex purified from as low as

4 9 106 HEK293 cells was sufficient for protein

identification by liquid chromatography tandem mass

spectrometry (LC-MS/MS). However, the authors

recommended starting with 3 9 107 cells for stan-

dard purification of protein complexes using the

proposed procedure. Recently, using this SBP-HA tag

and following a similar protocol, Bartoi et al. (2010)

and Wyler et al. (2011) successfully isolated GABAB

receptor complexes from transgenic mice and ribo-

somal subunit precursors from HEK 293 cells,

respectively.

SBP-His

Li et al. (2011) reported the efficient purification of

protein complexes from mammalian cells using a

SBP-His tandem tag. The authors initially tried the

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123

Interplay TAP system and cloned the target gene into

the pNTAP expression vector, obtaining a fusion

protein with N-terminal CBP-SBP tandem tag. How-

ever, whereas the fusion protein efficiently bound to

the streptavidin resin, it failed to bind to the

calmodulin resin. As mentioned in the above section,

the Interplay system has allowed successful purifica-

tion of various protein complexes. The failed binding

observed in this particular case could be due to

insufficient exposure of the CBP tag, which is at the

extreme N-terminus of the fusion protein. Switching

the tandem tag to the C-terminus may improve the

binding, as C-terminal tag generally has a lower

chance to be buried. However, considering that there

could be other factors affecting the efficiency of

calmodulin binding, Li et al. (2011) added an extra

His tag to the C-terminus of the target protein and

used immobilized metal-affinity chromatography

(IMAC) in place of CBP-calmodulin interaction

for the second round of purification (Fig. 2j). The

modified fusion protein efficiently bound to the

Ni-NTA resin and the captured protein complex

was eluted with 300 mM imidazole. More than 80%

of the target protein present in the biotin eluate from

the initial purification was recovered in the second

round of purification, putting the overall recovery of

the dual affinity purification at [50%, which is

among the highest of existing TAP tags. Starting with

eight 150-mm dishes of cells, Li et al. obtained a

decent amount of highly purified protein complex,

one-tenth of which was sufficient for protein identi-

fication by mass spectrometry.

Compared with Burckstummer’s method using

ProtG-SBP, His tag was used in place of ProtG to

allow more efficient binding of the target protein in

this latest approach. Furthermore, the protease cleav-

age step that caused yield loss had been avoided and

2 mM instead of 1 mM biotin was used to achieve

more complete elution from the streptavidin resin. All

these changes contribute to the higher protein recov-

ery achieved. In addition to the high yield, the SBP-

His combination has several other advantages. First,

both streptavidin and nickel resins are relatively

inexpensive and have a high capacity. For the

particular amount of starting material used in Li’s

study (i.e. eight 150-mm dishes of cells), 300 ll

streptavidin resin and 10 ll nickel resin were suffi-

cient for target protein recovery during the first

and second purifications, respectively. Second, the

sequential purification can be conveniently performed

in a single buffer system. Third, the IMAC approach

supports purification under denaturing conditions,

which is preferable for the isolation of in vivo cross-

linked protein complexes.

Stringency-tolerant tag

His-biotin

The His-biotin tag consists of one or two hexahisti-

dine and a biotinylation signal peptide (Fig. 2k), a

75-amino-acid sequence containing a specific lysine

residue that can be biotinylated in yeast and mamma-

lian cells by endogenous biotin ligase. Tagged proteins

can be sequentially purified by nickel and streptavidin

resins under fully denaturing conditions (Guerrero

et al. 2006; Tagwerker et al. 2006). In general, mild

purification conditions are preferred as they preserve

protein interactions and protein complex structure

(Seraphin et al. 2002). However, stringent conditions

can offer certain advantages. First, they prevent loss of

posttranslational modifications. For example, ubiqui-

tination, a sensitive modification that tends to be lost

under native conditions due to ubiquitin hydrolase

activity, is preserved under denaturing conditions.

Second, they are preferable for the purification of

cross-linked protein complexes. In vivo cross-linking

is an effective strategy for capturing weak and

transient interactions that are typically lost during

standard TAP procedures. However, cross-linking

may amplify the background, because nonspecifically

purified proteins can be cross-linked with other non-

relevant proteins. The stringent purification and wash

conditions compatible with the His-biotin tag can

effectively remove nonspecific interactions. It is

especially critical to perform the initial purification

using nickel resin when isolating His-biotin tagged

protein complexes from yeast, because compared with

mammalian cells yeast contains more endogenous

biotinylated proteins that can bind to the streptavidin

resin (Wang et al. 2007).

Localization and affinity purification (LAP) tag

GFP-S-peptide/His

In addition to single purpose TAP tags, Cheeseman

and Desai (2005) developed a dual functional tag,

Biotechnol Lett

123

which they referred to as the ‘‘localization and affinity

purification’’ (LAP) tag. The LAP tag contains GFP

coupled to either S-peptide (N-terminal tag) or His tag

(C-terminal tag) (Fig. 2l). GFP is used here as both

location indicator and the first purification tag. The

LAP-tagged fusion protein is first isolated using

protein A Sepharose with antibodies against GFP.

The captured protein is subsequently released by

treatment with TEV or human rhinovirus 3C (HRV3C)

protease. The protein is finally purified using either S

protein agarose or Ni-NTA, depending on the partic-

ular composition of the LAP tag. Recently, the LAP

tag was used for systematic characterization of human

protein complexes and allowed identification of novel

proteins that are involved in spindle assembly and

chromosome segregation (Hutchins et al. 2010).

Applications

Although the TAP strategy was originally developed

in yeast, it was quickly adapted to other systems

including insects (Forler et al. 2003), mammalian

cells (Cox et al. 2002), plants (Rivas et al. 2002) and

bacteria (Gully et al. 2003). Ideally, the tagged

protein should be expressed at physiological levels

and the endogenous untagged counterpart should be

suppressed. This is because that overexpression of the

tagged protein can result in isolation of large

quantities of chaperones and heat shock proteins,

and the untagged endogenous protein can compete

with for incorporation into protein complexes.

In yeast and bacteria, substitution of the target

gene with an allele encoding the tagged version of the

protein can be easily realized by homologous

recombination. In higher eukaryotic cells, however,

homologous recombination is not feasible in a high

throughput mode. Instead, maintaining physiological

levels of the tagged protein and depletion of the

untagged endogenous version is usually achieved by

stable transfection and RNA interference (RANi)

(Forler et al. 2003; Gregan et al. 2007), respectively.

Nevertheless, Poser et al. (2008) recently developed

the bacterial artificial chromosomes (BAC) tagging

approach that allows transgenes to be expressed in

cultured mammalian cells under the control of their

endogenous promoters and native regulatory ele-

ments. It is also possible to generate transgenic

(Angrand et al. 2006) or TAP-knockin mice (Zhou

et al. 2004; Fernandez et al. 2009). This is preferable

for the study of genes with more tightly regulated

expression. Furthermore, it enables characterization

of protein–protein interactions in a tissue specific

manner and allows the identification of novel inter-

acting partners that could be missed by studies using

cultured cells.

As a generic purification method, TAP enables

parallel characterization of multiple complexes. In

fact, TAP coupled with mass spectrometry has allowed

analysis of genome-wide protein–protein interactions

in yeast (Gavin et al. 2002; 2006; Krogan et al. 2006)

and bacteria (Butland et al. 2005; Hu et al. 2009;

Kuhner et al. 2009), and mapping of specific protein

networks/pathways in mammalian cells (Bouwmeest-

er et al. 2004; Brajenovic et al. 2004; Jeronimo et al.

2007) (Table 2).

Whereas the TAP method was developed for the

purification of noncovalent interactors, it has also

been used, with some modifications, to enrich

ubiquitylated proteins effectively from various sys-

tems (Mayor and Deshaies 2005; Saracco et al. 2009;

Golebiowski et al. 2010). In addition, TAP can also

be used to capture macromolecules other than

proteins. For instance, Nonne et al. (2010) recently

described a TAP-based approach that allows specific

pull down of mRNA targets of miRNA.

Conclusions

The introduction of TAP represents a major improve-

ment in isolation of in vivo formed protein complex.

The high sample purity conferred by the sequential

purification greatly simplifies subsequent identifica-

tion and validation of the isolated proteins as true

interacting partners. TAP coupled with mass spec-

trometry has emerged as a powerful tool to delineate

protein complexes involved in various biological

processes. However, despite its strength, the original

TAP approach has several limitations (Puig et al.

2001; Volkel et al. 2010). First, fusion of the 21-kDa

ProtA-CBP tag may cause loss of protein function or

disturb protein complex assembly, and sometimes the

tag is not sufficiently exposed to allow binding to the

affinity matrix. Second, yields from non-yeast sys-

tems are relatively low and consequently large

amount of cells are required as starting material.

Third, weak and transient interactions are typically

lost during standard procedures.

Biotechnol Lett

123

In general, tag-specific problems (e.g., structure/

function disruption, variable exposure, steric exclu-

sion, etc.) can be solved or minimized by using

smaller tags (e.g., FLAG-HA and SBP-HA) and/or

attaching the tag at different ends. Cipak et al. (2009)

found that adding a flexible linker between the TAP

tag and the target protein can also effectively

minimize the tag-induced negative impact. The use

of several alternative TAP tags, as introduced above,

has shown to significantly improve protein yields in

non-yeast systems. These high-recovery tags are

especially useful for purification of protein com-

plexes from difficult-to-cultivate cells (e.g., neuronal

and immune cells). Finally, TAP coupled with in vivo

cross-linking, which freezes all types of protein

interactions as they occur in the cell, has the potential

to identify weak and transient interacting partners

(Tardiff et al. 2007; Woodcock et al. 2009). The His-

biotin tag is especially suitable for this approach

because the stringent conditions it supports can

significantly reduce cross-linking introduced nonspe-

cific background.

Although none of the newly developed TAP tags

has gained the same popularity as the original ProtA-

CBP tag, which remains predominantly used in every

system (Li 2010), they nevertheless make a signifi-

cant contribution towards improving the feasibility/

efficiency of the TAP methodology by providing

alternative options.

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