Send Orders of Reprints at email@example.com
8 Current Chemical Genomics, 2012, 6, (Suppl 1-M2) 8-17
1875-3973/12 2012 Bentham Open
The HaloTag: Improving Soluble Expression and Applications in Protein
Scott N. Peterson and Keehwan Kwon*
J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, Maryland 20850, USA
Abstract: Technological and methodological advances have been critical for the rapidly evolving field of proteomics. The
development of fusion tag systems is essential for purification and analysis of recombinant proteins. The HaloTag is a 34
KDa monomeric protein derived from a bacterial haloalkane dehalogenase. The majority of fusion tags in use today utilize
a reversible binding interaction with a specific ligand. The HaloTag system is unique in that it forms a covalent linkage to
its chloroalkane ligand. This linkage permits attachment of the HaloTag to a variety of functional reporters, which can be
used to label and immobilize recombinant proteins. The success rate for HaloTag expression of soluble proteins is very
high and comparable to maltose binding protein (MBP) tag. Furthermore, cleavage of the HaloTag does not result in pro-
tein insolubility that often is observed with the MBP tag. In the present report, we describe applications of the HaloTag
system in our ongoing investigation of protein-protein interactions of the Y. pestis Type 3 secretion system on a custom
protein microarray. We also describe the utilization of affinity purification/mass spectroscopy (AP/MS) to evaluate the
utility of the Halo Tag system to characterize DNA binding activity and protein specificity.
Keywords: HaloTag, protein-protein interactions, protein-DNA interactions, expression, immobilization, Type 3 secretion fac-
tors, E. coli RpoA.
sharply over the past 15 years . These advances have en-
abled the sequencing of many large and small genomes, re-
sulting in over 3,000 bacterial genomes including ~150 ar-
chaea and nearly 200 eukaryotic and mammalian genome
sequences (http://www.ncbi.nlm.nih.gov/sites/genome) to be
completed. The access to this massive quantity of data has
had a strong ripple effect leading to an increased demand for
new technologies that will enable scientists to study the ac-
tivities and functions of these gene sequences in a high
throughput manner. Among the numerous discoveries en-
abled by genome sequence data, one somewhat unanticipated
finding relates to the fact that at least one-third of the open
reading frames (ORFs) encoded in genomes has no predicted
function based on BLAST analysis [2-4]. Interestingly, the
number of genes of unknown function increases in a linear
manner as we sequence additional genomes . One might
imagine that as we sequence more genomes, the rate that
novel genes are identified would begin to decrease rapidly.
This is clearly not the case though and strongly support the
view that the number of unique gene sequences and func-
tions encoded on our planet is very large. For most microbial
species, 10-30% or more of the ORFs encoded in one
strain’s genome are novel compared to another strain belong-
ing to the same species. The gene pool of many bacterial
species may exceed several tens of thousands of unique
Advances in DNA sequencing technology have increased
*Address correspondence to this author at the J. Craig Venter Institute, 9704
Medical Center Drive, Rockville, Maryland 20850: Tel: 301-795-7647;
Fax: (301)294-3142; E-mail: firstname.lastname@example.org
genes. It is likely that by the end of this decade, we will have
sequenced over 10 million genes of unknown function!
This humbling realization emphasizes the need for sub-
stantial improvements in the area of functional genomics if
we are to keep pace with the ever-increasing ease that genes
and genomes are sequenced. One phenomenon that have
been documented, referred to as non-orthologous gene dis-
placement (NODs) may provide an inroad to tackling the
monumental problem of determining the function of unchar-
acterized genes. NODs represent cases where two proteins
perform the same cellular function but do not possess an
ancestral relationship. We know of several cases like eu-
karyotic and prokaryotic DNA polymerases that essentially
carry out the same cellular functions, but do not share com-
mon ancestral relationships. In other words these functions
evolved independently during evolution. The vast majority
of the assigned functions of genes are based on BLAST and
orthology (conservation of DNA or amino acid sequence). If
genes arise independently they by definition do not share
ancestry nor do they share amino acid sequence identity. The
scientific research community has developed strategies to
assay a wide range of known protein functions over the
years, it may follow that the screening of novel proteins of
unknown function using familiar assay systems will yield a
surprising number of experimentally determined gene func-
tions. While this explanation may partially explain the rea-
son we are accumulating more and more genes of unknown
function in our databases, we remain highly ignorant as to
the frequency of NODs in nature.
such as microfluidics and DNA and protein microarrays,
Massively parallel technologies have been developed,
Improving Soluble Expression and Applications in Protein Functional Analysis Current Chemical Genomics, 2012, Volume 6 9
which present important vehicles to partially enable the
large-scale characterization of gene/protein function [6-12].
Our ability to determine the function of genes places strong
demands on a variety of disciplines related to recombinant
protein technologies. The large-scale characterization of pro-
tein function requires very efficient recombinant proteins
production in a high-throughput environment and the neces-
sary automation to perform high-throughput functional
screens [13, 14]. Likewise, complementary technologies that
broaden the use of recombinant proteins such as labeling
methods, sub-cellular localization determination, enzymatic
activity and substrate specificity will also need to be devel-
oped and advanced if we are to make significant progress.
Among the numerous challenges associated with large-
scale functional characterization of proteins is the choice of
expression systems that are to be employed. Given the fact
that several systems offer some discrete advantage, in an
ideal world, one would employ many platforms. For practi-
cal reasons researchers are forced to make difficult decisions
regarding which platform provides the greatest overall utility
for the objectives in question. Among the variety of tools
being developed that show promise of enabling the func-
tional characterization of protein function, the HaloTag tech-
nology developed by scientists at Promega (Madison, WI) is
notable [15, 16]. Here we provide an overview of functional
assays and experience we have developed in conjunction
with the HaloTag technology.
We have used the HaloTag technology for a number of
functional studies, including protein microarrays, affinity
purification of DNA-protein, protein-protein interactions,
and protein complex identification [7, 17]. The HaloTag is a
modified haloalkane dehalogenase designed to covalently
bind a series of chloroalkane derivatives such as fluoro-
phore-labeled ligands (Promega). We have observed im-
proved solubility of fusion proteins using this system, com-
parable to that achieved by the best solubilization fusion
partner, the maltose-binding protein MBP . The HaloTag
vector (Promega) adopted a Flexi cloning system that uses
traditional restriction site cloning methods. We found this
cloning method to be inadequate for high-throughput cloning
of genes, and have adapted the cloning platform for com-
patibility with Gateway and Ligation Independent Cloning
(LIC) procedures [19-22]. We have used these vectors in a
number of studies including the expression and purification
of proteins derived from Influenza virus H1N1, Y. pestis, S.
pneumoniae and B. mallei. Genes were expressed using sev-
eral expression systems including E. coli, a cell-free (wheat
germ) system and mammalian cells. The HaloTag supports
development of functional assays, such as fluorescence po-
larization, FRET, on-chip purification in protein microarrays
and also allows monitoring sub-cellular protein localization.
The rapid covalent attachment of the HaloTag to its specific
ligand is a critical feature that separates the HaloTag from
any other tags that use reversible interactions . The high
affinity covalent interaction is extremely rapid and allows
binding reactions to be carried out in minutes. This has prov-
en advantageous in that we observe a dramatic reduction in
the background, non-specific binding events that reduce sig-
nal to noise assay ratios [16, 24].
HISTORICAL DEVELOPMENT OF HALOTAG
engineering of a bacterially encoded haloalkane dehydro-
genase (DhaA) derived from a Rhodococcus spp , carried
out in the laboratories at Promega [15, 16, 24]. The occur-
rence of this enzyme is phylogenetically restricted to a small
number of taxa. The 34 kDa protein cleaves at the carbon-
halogen bond of a number of aliphatic halogenated com-
pounds through a mechanism involving a hydrolytic triad
within the active site of the enzyme. During the carbon-
halogen cleavage reaction, the enzyme forms a transient co-
valent complex with its substrate, leading to the nucleophilic
displacement of the terminal halogen using Asp106. The
complex is hydrolyzed in a reaction involving His273
through the activation of a water molecule. In order to stabi-
lize this intermediate, the His273 residue was replaced with a
Phe residue that occupies a similar volume in space but does
not have the potential as a base to carry out the hydrolysis
reaction. Therefore, the covalently linked substrate remains
trapped in the active site of the enzyme. Mutagenesis of
some residues was made to increase the accessibility of the
ligand for the active site and for others to enhance solubility
and additional characteristics in the final HaloTag protein.
These efforts have resulted in the provision of a novel and
robust system for conducting recombinant protein studies in
a wide variety of formats.
The development of the HaloTag is the result of rational
POTENTIAL ADVANTAGES OF COVALENT LINK-
tional characterization of proteins have led to the develop-
ment of a variety of novel methodological strategies and
technologies. Many of these strategies rely on the immobili-
zation of recombinant proteins to matrices with a very large
surface area [9, 11, 26-31]. In this regard many of the bio-
chemistry or physical interaction studies being carried out
are associated with unique challenges presented by large-
scale screening and the immobilization to solid substrates
that in some cases may generate significant non-specific
binding and high levels of background in the assays per-
formed. The HaloTag technology offers some discrete and
potentially important advantages to address these two issues
based on the covalent and very high affinity interaction be-
tween the HaloTag and its ligand [15, 16, 32]. The covalent
linkage of the HaloTag to immobilized surfaces ensures that
high stringency washes may be performed without concern
of removing the immobilized proteins . Perhaps equally
important is the high affinity interaction of the HaloTag and
its ligand. The on rate of the interaction at typical protein
ligand concentrations drives the reaction to near completion
very rapidly. In this regard, the functional assays performed
with HaloTag recombinant proteins can be conducted in a
reduced time frame; thereby decreasing the mass-action,
non-specific background signals that may be facilitated by
longer incubation times.
Research objectives focused on high throughput func-
ADAPTATION OF HALOTAG TO GATEWAY EX-
production and functional screening is the selection of an
One of the essential elements for high-throughput protein
10 Current Chemical Genomics, 2012, Volume 6 Peterson and Kwon
expression vector with a specific fusion tag. The trends in
high-throughput recombinant protein expression indicate that
no single expression system is ideal for all target proteins.
Therefore, many expression pipelines include multiple ex-
pression vectors which are used in parallel to increase the
overall success rate of recovering soluble proteins. However,
in order to use multiple expression vectors, efficient cloning
methods such as the Gateway recombination cloning method
are required [19, 20]. Although the use of multiple expres-
sion vectors increases the number of recovered soluble target
proteins, for practical purposes, the, use of expression vec-
tors is often limited to one or a few vectors in most high
throughput gene cloning pipelines. Therefore, an ideal ex-
pression vector possesses excellent fusion tag properties
(solubility and purification efficiency) and a high throughput
cloning procedure amenable to automation. We have at-
tempted to strike this ideal by constructing a series of ex-
pression vectors that merge the qualities associated with the
HaloTag to the ease and efficiency associated with the either
LIC or Gateway cloning methods. The Gateway compatible
expression vector has the added advantage that it allows in-
vestigators to utilize existing entry clone sets which have
been produced and made available through public reposito-
ries (http://www.beiresources.org) [14, 18]. We have evalu-
ated the outcomes of a number of protein expression trials
using these chimeric vectors.
The vectors, pFN18A, pFN19A, pFC20A and pFC14A
were obtained from Promega for expression of various target
proteins in E. coli, cell-free lysates and mammalian expres-
sion systems (Fig. 1). We modified these vectors in a variety
of ways. Each of the modified vectors contains the E. coli
ccdB cassette which encodes a product that is toxic to E. coli
. We adapted the Gateway cloning method to prepare
clones which were easier to use than existing entry clones.
The expression vector, pGW-nHalo, is based on the vector
pFN18A which replaced the barnase with the attR recombi-
nation cloning sites and ccdB cassette. We also constructed
pHis-cHalo another Gateway compatible vector based on
pFN20A and T02 (pHis) vectors  that contains an N-
terminal His-tag and a C-terminal HaloTag. We also con-
structed a ligation independent cloning vector with a C-
terminal HaloTag (pLIC-Halo) based on the pMCSG7 vector
backbone  and consists of an N-terminal His-tag and a
C-terminal HaloTag. The His-tag can be removed by throm-
bin cleavage after purification [21, 22]. The addition of the
His-tag in the vectors enables the use of the His-tag for puri-
fication, when down-stream applications of the purified pro-
tein require the HaloTag for fluorophore labeling.
COMPARISON OF EXPRESSION VECTORS
teins using the various HaloTag vectors (Fig. 1) were evalu-
ated in E. coli, cell-free expression system and mammalian
cells and compared with previous expression studies that
employed fusion proteins such as: His-tag, MBP, DsbA and
GST (Table 1 and Supplementary Table 1) [14, 18]. As de-
picted in Fig. (1), each HaloTag vector has specific charac-
teristics such as the location of the HaloTag, drug resistance
markers and cloning strategies. Four of those vectors,
pFN19A, pFC20A, pHis-cHalo and pLIC-Halo all contain
dual promoters, T7 and SP6, which express proteins in either
E. coli or wheat germ in vitro expression systems. As a con-
trast, vectors, pFN18A and pGW-nHalo, allow the expres-
sion of proteins in E. coli expression system with the T7
The His-tag expression vector, T02 (pHis) yielded solu-
ble proteins in 43.2 % of attempts when targeting the com-
plete set of ORFs encoded in S. pneumoniae TIGR4 . A
second study focused on expression of proteases resulted in
similar outcomes with 39.6% success . The success fre-
quencies were below 50% for each of the vectors tested in
these studies except cases employing the MBP-tag or the
HaloTag. The pMBP produced soluble proteins for more
than 70% of target proteins. Both the pFN19A, and the
pGW-nHalo, which are N-terminal HaloTag vectors, pro-
duced soluble proteins in E. coli at very similar frequencies.
Success rates in recovering solubly expressed target pro-
pFN19A (T7, SP6)
Fig. (1). The HaloTag expression vectors used for protein expression and functional studies.
Various expression vector systems were used for HaloTag recombinant protein expression. The vectors, pFN18A, pFN19A, pFC20A and
pFC14A were obtained from Promega for expression of target proteins in E. coli, cell-free and mammalian expression systems. In order to
modify applicable cloning methods, we further modified these vectors to contain the ccdB cassette for positive selection of cloned plasmids.
The expression vector, pGW-nHalo is based on pFN18A and ccdB cassette was incorporated into the vector. The pHis-cHalo was based on
pFN20A and T02 (pHis) vector . The expression vector, pLIC-Halo was also based on pFC20A and LIC cloning site was incorporated
with ccdB cassette. These vectors were used for expression and solubility studies of proteins in S. pneumoniae TIGR4, Y. pestis KIM 10, B.
mallei ATCC 23344 and H1N, and for functional analysis.
Improving Soluble Expression and Applications in Protein Functional Analysis Current Chemical Genomics, 2012, Volume 6 11
Table 1. Comparison of Success Rates of Soluble Expression of Recombinant Proteins which Derived from Various Expression
Fusion tag Expression Solubility HaloTag Vector Expression Solubility
pHis: His-tag1 59.5% 43.2% pFN18A3 (23) 73.9% 56.5%
pHis: His-tag2 54.0% 39.6% pGW-nHalo3 (74) 82.4% 70.3%
pMBP: ΔSP-MBP2 72.7% 70.1% pFN19A3 (52) 75.0% 69.2%
pSP-MBP: MBP2 64.7% 43.9% pFC203 (67) 67.2% 61.2%
pDsbA: DsbA2 58.8% 47.6% pFC14A4 (10) 80.0% N/A
pEXP7: GST2 49.7% 42.8% HaloTag (average) 75.2% 65.7%
1Genome-wide protein expression and purification of S. pneumoniae proteome success rates were calculated based on efforts applied to 1529 destination
clones . 2Putative proteases derived from S. pneumoniae TIGR4, B. anthracis Ames and Y. pestis KIM. Success rates were calculated based on 187 destina-
tion clones . 3Combination of protein sets (23-80 clones) of DNA binding proteins, Type 3 secretion system, Type 6 secretion system and/or randomly
selected proteins in E. coli, S, pneumoniae TIGR4, Y. pestis B. anthracis, Ames, and Burkholderia mallei ATCC23344. 410 H1N1 proteins were used in the
study and solubility information is not available. The numbers in parentheses are the number of clones in the study.
Our efforts pertaining to the construction of a vector (pHis-
cHalo) containing the Gateway attR cloning sites and a C-
terminal HaloTag was not generally useful for protein ex-
pression for reasons that remain unclear, while pGW-nHalo,
Gateway compatible vector with an N-terminal HaloTag,
displayed excellent expression and solubility of target pro-
teins, similar to outcomes obtained with pFN19A that also
contains an N-terminal HaloTag. Influenza virus (H1N1)
proteins were expressed using pFC14A, which contains the
CMV promoter and a C-terminal HaloTag, and 8 proteins
from this virus were well expressed in HEK293T. These
same proteins were expressed in truncated form when using
the E. coli expression system. Although target proteins for
the expression attempts are not identical and therefore not
directly comparable, the proteins in attempts using HaloTag
vectors contain a randomly selected set and difficult mem-
brane localized protein sets such as type III and type VI se-
cretion systems. Overall, the body of experience using Halo-
Tag is now large enough to enable comparison to overall
outcomes associated with other vector systems and conclude
that the HaloTag enhances expression and solubility of target
proteins to levels comparable to that of the previously de-
fined “best” solubilization tag, MBP [36, 37].
INCREASE SUCCESS RATES OF SOLUBLE EX-
pression and purification of proteins are essential. Here, we
describe two strategies we employed to increase the success
rate of soluble expression/purification of proteins of interest.
First, a complementary pair of expression vectors containing
the same fusion tag (C-terminal and N-terminal) increases
the overall recovery of soluble proteins. We have used the
expression vectors, pFN19A and pFC20A for this purpose to
express a group of E. coli proteins (Fig. 2). Second, we eval-
uated the success rate of traditional column-based purifica-
tion procedures to in situ purification and determined that the
latter increased overall success and yield of purified proteins
In order to characterize proteins of interest, soluble ex-
Fig. (2). Expression of E. coli proteins for protein-protein interactions. The HaloTag recombinant proteins were visualized with TMR ligand.
12 Current Chemical Genomics, 2012, Volume 6 Peterson and Kwon
Fig. (3). A comparison of 20 proteins derived from S. pneumoniae using different expression and purification schemes. Manual in situ purifi-
cation of E. coli, in vitro expressed proteins and column purification were compared. The purified proteins were visualized using rabbit anti-
HaloTag antibody followed by a goat anti-rabbit antibody conjugated to the dye Alexa555 (upper). The purity of the proteins recovered from
each strategy was examined by comparing the ratio of signal generated by an anti-E. coli antibody to that of anti-HaloTag antibody. Each
well represents purification of the following protein, 1: SP_0291, 2: SP_0308, 3: SP_0321, 4: SP_0435, 5: SP_0604, 6: SP_0845, 7:
SP_0954, 8: SP_0979, 9: SP_1102, 10: SP_1504, 11: SP_1572, 12: SP_1631, 13: SP_1650, 14: SP_1671, 15: SP_1699, 16: SP_1752, 17:
SP_1802, 18: SP_1925, 19: SP_1959, and 20: SP_2209.
(C-terminal HaloTag) to increase the overall recovery of
soluble proteins of E. coli proteins of interest, LeuC, LeuD,
HisF, HisH, RpoA, RpoB, GyrA and GyrB. For these studies
we used two E. coli expression strains to enhance the recov-
ery of soluble proteins. BL21(DE3)/pMagic, an E. coli B
strain derivative containing the pMagic plasmid that encodes
tRNAs that are rare in E. coli and KRX/pGro7, a K-12 de-
rivative containing a plasmid expressing the chaperone com-
plex, GroEL/ES . The use of pFN19A and pFC20A vec-
tors displayed similar outcomes in most cases but also dis-
played complementary outcomes in several instances as
shown in Fig. (2). For example, LeuD and GyrA displayed
higher soluble expression using pFC20A while almost no
soluble protein was recovered with pFN19A. In contrast,
HisF, HisH and RpoB were recovered as soluble proteins
only pFN19A. Similarly, HisF and GyrB were expressed in
soluble form at higher levels in vector pFN19A in
KRX/pGro7 while soluble LeuC was expressed at higher
levels using BL21(DE3)/pMagic. Soluble HisF was obtained
solely with N-terminal HaloTag vector in KRX/pGro7. The
combination of expression vectors, pF19A and pFC20A and
two expression strains allowed the recovery of all targets in
soluble form with adequate yield and purity.
As part of our ongoing efforts to compare a variety of
strategies for recombinant protein expression and purifica-
tion to determine whether any provide a means for achieving
higher overall success frequencies in the recovery of soluble
recombinant protein. We exploited the covalent linkage of
HaloTag recombinant proteins as a means of performing
direct protein purification from crude E. coli lysates or from
in vitro expression extracts using HaloLink microarray slides
(Fig. 3). We randomly selected 20 ORFs encoded in the ge-
We used pFN19A (N-terminal HaloTag) and pFC20A
nome of S. pneumoniae and cloned these sequences into
pFC20A. Recombinant proteins were either expressed in the
BL21(DE3)/pMagic strain or by in vitro expression using the
TnT® SP6 Coupled Wheat Germ Extract System (Promega).
The over-expressed proteins derived from BL21(DE3)/
pMagic were purified using either HaloLink resin resulting
in recovery of 75% of targets as soluble protein. When these
proteins were expressed and purified using direct purifica-
tion on HaloLink glass slides we recovered 100% of the tar-
get proteins in soluble form. Finally, when using in vitro
transcription and translation systems followed by direct puri-
fication using HaloLink slides we recovered 85% of the tar-
get proteins in soluble form. Conclusions drawn from these
studies must be taken with caution, however it appears that
direct purification of recombinant proteins whether ex-
pressed in vitro or in E. coli may be more successful than
traditional column-based purification schemes. The average
purity of recovered proteins over-expressed in the E. coli
BL21(DE3)/pMagic strain and purified using in situ purifica-
tion is estimated to be more than 90% which is adequate for
a variety of downstream applications.
TO USE OF HALOTAG RECOMBINANT PROTEINS
IDENTIFY PROTEIN-PROTEIN INTERACTIONS
we see that few proteins operate in isolation of other macro-
molecules, particularly other proteins. The two-hybrid meth-
od and immunoprecipitation “pull down” experiments have
contributed to our growing perception that proteins often
function via physical interaction with one or more proteins
[12, 39]. Our knowledge of numerous binary interactions
between proteins and multi-protein complexes e.g. RNA and
DNA polymerase, ribosomal subunits etc is extensive for
As we learn more about the cellular functions of proteins
Improving Soluble Expression and Applications in Protein Functional Analysis Current Chemical Genomics, 2012, Volume 6 13
these examples but fundamentally lacking in others. Inde-
pendent methods are needed to validate and discover protein-
protein interactions . We have used the HaloTag tech-
nology in a number of formats as a means of identifying or
validating a number of binary protein interactions and also to
id ntify constituents of multi-protein complexes [7, 40].
Protein interactions that occur within the Y. pestis Type 3
secretion system (T3SS) were identified using a protein ar-
ray-based method in which the labeled HaloTag recombinant
proteins were used as prey to detect binary protein interac-
tions with immobilized bait proteins. The T3SS apparatus,
also known as an injectisome, functions to directly inject
effector proteins expressed by the bacterium into its mam-
malian host during infection [41-45]. To carry out this inter-
rogation we cloned the bait proteins (T3SS) into pMBP (His-
MBP tag) previously reported in , that were immobilized
to a Cu2+ coated microarray slide surface (Fig. 4) . The
immobilized bait proteins were challenged with specific
HaloTag prey proteins which were derived from pFN18A to
establish the specificity of their interactions using indirect
detection via an anti-HaloTag antibody or Biotin labeled
HaloTag followed by fluorescently labeled streptavidin. The
pFN18A vector was used for this study because the HaloTag
recombinant T3SS proteins derived from pFN19A and
pFC20A were partially degraded when expressed in E. coli.
These experiments are particularly challenging since the
T3SS is a multi-protein complex involving a number of
membrane localized components that are difficult to express
as soluble proteins. An example of the results achieved using
this strategy is shown in Fig. (4). In this instance, when Hal-
ds for determining the interaction of
oTag prey protein Y0049 (LcrG) is used to interrogate the
protein microarray it interacts specifically with Y0050
(LcrV), an interaction that has been reported previously us-
ing independent metho
th se proteins [47-51].
We evaluated the use of HaloTag in a more challenging
goal to capture the subunits of multi-protein complexes. We
selected a well-characterized multi-protein complex, RNA
polymerase from E. coli to examine the pull down scheme
wherein one suspected member of a protein complex is fused
to HaloTag. Based on the work of several studies it is known
that RpoA forms direct contacts with itself, AceE, RplA,
RpoC, NusA and RpoB, whereas indirect linkages within the
complex include the additional proteins TufA and Tig [52-
58]. We cloned and over-expressed the RpoA subunit as an
N-terminal HaloTag (pFN19A) fusion protein in E. coli,
BL21(DE3)/pMagic. The assumption made in this experi-
mental procedure is that the fusion protein will retain its abil-
ity to interact with the other proteins in the complex with
relatively similar efficiency as the endogenously expressed
RpoA. The RpoA in the pFN19A vector was over-expressed
in 5 mL E. coli culture. The RpoA derived from the whole
cell lysate was immobilized onto HaloLink resin and washed
extensively to eliminate non-specific interacting proteins.
Following recovery of the fusion protein, several protein
bands were recovered (Fig. 5A). These bands were cut from
the gel and subjected to MALDI-TOF/TOF-MS to identify
those proteins present in the RpoA complex. Our results il-
lustrate the power of the approach as all of the known mem-
bers of the protein complex were recovered as shown in Fig.
Fig. (4). Identification of T3SS Interactions in situ Using Protein Microarrays. (A) Scheme used to identify protein-protein interaction using
HaloTag recombinant proteins and His-MBP tagged recombinant proteins. (B) Immobilized His-MBP tagged T3SSs on Cu2+/IDA/PEG were
visualized with anti-His-tag antibody (left) and interacting proteins with IcrG were detected by the rabbit anti-Halo antibody and goat anti-
rabbit antibody labeled with Alexa555 (right).
14 Current Chemical Genomics, 2012, Volume 6 Peterson and Kwon
5B. This platform can be easily adapted to high throughput
platform such as a 96-well format, thus allowing AP/MS to
be performed in a high throughput manner.
s that require phos-
phorylation for DNA binding activity.
USE OF HALOTAG RECOMBINANT PROTE
IDENTIFY PROTEIN-DNA INTERACTIONS
transcriptional regulatory proteins has been significant for
nearly three decades now. There are a variety of methods for
studying these interactions but the majority of these are re-
fractory to high throughput characterization. We have evalu-
ated a number of methods including gel mobility shift as-
says, fluorescence polarization, ChIP-chip and ChIP-Seq
analysis and others [8, 10, 59-70]. Each approach has spe-
The interest in DNA protein interactions, particularly of
cific advantages and disadvantages with respect to ease, re-
producibility, sensitivity and specificity. The proteomic pro-
filing of transcription factors is often hampered by the low-
level expression of these proteins preventing their visualiza-
tion on 2DE MS/MS based experiments or LC/MS/MS stud-
ies. We enriched these proteins from crude lysates derived
from Y. pestis by passing the lysate over a DNA cellulose
column. The eluted proteins were indeed strongly enriched
for transcription factors and other nucleic acid binding pro-
teins. Among the list of recovered proteins was a set of 16
hypothetical proteins. We wished to establish whether these
genes of unknown function represented a new class o
sc iption factors or nucleic acid binding proteins.
We developed an approach to evaluate the DNA binding
activity and specificity of these proteins as described below.
In this scheme, we cloned each of the putative transcription
factors into pFN19A N-terminal HaloTag expression vector.
The recombinant proteins were expressed in BL21(DE3)/
pMagic. These proteins were then immobilized onto Halo-
Link slides. Among the 16 Y. pestis target proteins 12 were
expressed in E. coli and 10 of these were recovered as solu-
ble protein. Nine of the soluble proteins were effectively
purified by direct purification on HaloLink slides (Fig. 6).
We next fluorescently labeled sheared Y. pestis genomic
DNA with Cy5. The labeled genomic DNAs were then
mixed with each immobilized HaloTag fusion protein in ei-
ther low or high salt buffer to allow DNA-protein interac-
tions to occur. After appropriate washing of the slide surface,
the bound genomic DNA is recovered from the array and
used as a hybridization probe of a second DNA oligonucleo-
tide tiling microarray. This microarray represents the entire
Y. pestis genome as a series of overlapping 60-mer oligonu-
cleotides alternately covering each strand of DNA and al-
lows the approximation and partial identification of the spe-
cific DNA sequences bound by the transcription factor. This
straight-forward method is amenable to moderate throughput
but can be envisioned as a means of characterizing all anno-
tated transcription factors encoded in a genome of interest.
While our experience with this strategy is still limited it is
anticipated that the method success will be linked to the af-
finity of the protein for its cognate DNA sequence motifs
and further by our ability to capture growth conditions that
permit expression of transcription factors such that they are
activated for specific DNA binding such as is expected for
the case of two-component regulator
Fig. (5). Multi-protein Complex Discovery. (A) The pull-down
study using HaloTag recombinant RpoA. M: molecular weight
marker; 1: unbound protein; 2: wash; 3: eluted protein after TEV
protease cleavage, 4: eluted protein after removal of TEV protease;
5: concentrated protein sample. The arrows indicate the position of
HaloTag recombinant (lane 1) and cleaved (lane 3) RpoA. (B) In-
teraction map of E. coli proteins which identified by MALDI-
MS/MS from the E. coli RpoA pull-down study.
tein production platforms and examined the enhancement of
soluble expression of the proteins of interest. We also exam-
ined the use of the HaloTag to high throughput functional
studies such as protein-protein interactions and protein-DNA
interactions. Several vectors containing HaloTag were made
compatible with high throughput cloning strategies and ex-
amined for their efficiency in expressing soluble protein. The
N-terminal HaloTag Gateway vector (pGW-nHalo) showed
that the HaloTag recombinant proteins were solubly ex-
pressed with a high success rate and can be used for high
throughput cloning using existing entry clone sets. Soluble
expression attempts of proteins of interest in E. coli, in vitro
We have adapted the HaloTag technology to current pro-
Improving Soluble Expression and Applications in Protein Functional Analysis Current Chemical Genomics, 2012, Volume 6 15
Fig. (6). Protein Microarray and DNA Tiling Microarray to Identify Protein-DNA Interactions. of hypothetical proteins in Y. pestis KIM.
Binding of Cy5 labeled sheared genomic DNA onto a 16-pad protein array (A) in low salt binding buffer (25 mM KCl) and (B) in high salt
buffer (150 mM KCl). (C) Array image after recovery of the bound DNAs. (D) DNA tiling microarray with the recovered DNA from protein
array. The color represents the amount of DNA bound to the proteins. The color scale represents the strongest signals as red followed by
orange, yellow, green, blue and black.
and mammalian expression systems were conducted using
various HaloTag vectors and the results demonstrated overall
high success rates. A combination of N-terminal and C-
terminal HaloTag vectors increases overall success rate of
soluble protein recovery. We have employed the HaloTag
technology in other contexts using protein microarrays for
high throughput assay for anti-sera screening and other pro-
tein functional analysis. In the protein array schemes, the
HaloTag recombinant proteins were successfully used as
prey proteins for identification of protein-protein interactions
in Y. pestis T3SS with other fusion tagged recombinant pro-
teins, and as bait proteins to identify DNA binding activity
of hypothetical proteins. The HaloTag was successfully used
for pull-down assays involving E. coli RpoA as part of a
multi-protein complex. While we describe here only a lim-
ited number of applications of the HaloTag technology,
many more strategies are enabled by this versatile technol-
ogy. In these early days of the post-genomic era, HaloTag
and other technologies will be important vehicles for better
understanding the breadth of protein functions encoded by
the awe inspiring number of unique proteins encoded on our
itute of Health, under contract No. N01-
e on the publisher’s website along with the published
onfirm that this article content has no con-
The authors c
flicts of interest.
 C. Next-generation se-
pective on the genetic content of the alkaliphilic
haloarchaeon Natrialba magadii ATCC 43099T. BMC Genomics
2012; 13(1): 165.
Kasuga T, Mannhaupt G, Gl
logenetic distribution and genomic features in Neurospora crassa.
PLoS One 2009; 4(4): e5286.
Clamp M, Fry B, Kamal M, et
and noncoding genes in the human genome. Proc Natl Acad Sci
USA 2007; 104(49): 19428-33.
Raskin DM, Seshadri R, Pukatzki SU, Mekalanos JJ. Bacterial
genomics and pathogen evolution. Cell 2006; 124(4): 703-14.
Fordyce PM, Gerber D, Tran D, et al. De novo identification and
with microfluidic affinity analysis. Nat Biotechnol 2010; 28(9):
Hurst R, Hook B, Slater MR, Hartnett J, Storts DR, Nath N. Pro-
strategies on signal-to-background ratios. Anal Biochem 2009;
Johnson DS, Mortazav
mapping of in vivo protein-DNA interactions. Science 2007;
Phizicky E, Bastiaens PI, Zhu H, Snyder M, Fields S. Protein anal-
ysis on a proteomic scale. Nature 2003; 422(6928): 208-15.
Pugh BF, Gilm
interactions in living cells. Genome Biol 2001; 2(4): RE-
Zhu H, Bilgin M, Bangham R, et al. Global analysis of protein
activities using proteome chips. Science 2001; 293(5537): 2101-5.
Zhang L, Villa NY, Rahman MM, et al. Analysi
 ass NL. Relationship between phy-
al. Distinguishing protein-coding
acterization of transcription-factor binding sites
raction studies on protein arrays: effect of detection
 i A, Myers RM, Wold B. Genome-wide
 our DS. Genome-wide analysis of protein-DNA
 s of vaccinia virus-
host protein-protein interactions: validations of yeast two-hybrid
screenings. J Proteome Res 2009; 8(9): 4311-8.
review of the manuscript. We also thank Sarah Grimshaw for
proofreading of the manuscript. This work was supported by
the National Institute of Allergy and Infectious Diseases,
We thank Dr. Marjeta Urh for helpful discussion and
S PPLEMENTARY MATERIAL
Supplementary material (Supplementary Table 1.pdf) is
CONFLICT OF INTEREST
Berglund EC, Kiialainen A, Syvanen A
quencing technologies and applications for human genetic history
and forensics. Investig Genet 2011; 2: 23.
Siddaramappa S, Challacombe JF, De Castro RE, et al. A compara-
tive genomics pers
16 Current Chemical Genomics, 2012, Volume 6 Peterson and Kwon
 Lesley SA. High-throughput proteomics: protein expression and
purification in the postgenomic world. Protein Expr Purif 2001;
Kwon K, Pieper R, Shallom S, et al. A correlation analysis of pro-
tein characteristics associated with genome-wide high throughput
expression and solubility of Streptococcus pneumoniae proteins.
Protein Expr Purif 2007; 55(2): 368-78.
Los GV, Encell LP, McDougall MG, et al. HaloTag: a novel pro-
tein labeling technology for cell imaging and protein analysis. ACS
Chem Biol 2008; 3(6): 373-82.
Ohana RF, Encell LP, Zhao K, et al. HaloTag7: a genetically engi-
neered tag that enhances bacterial expression of soluble proteins
and improves protein purification. Protein Expr Purif 2009; 68(1):
Gallo S, Beugnet A, Biffo S. Tagging of functional ribosomes in
living cells by HaloTag(R) technology. In Vitro Cell Dev Biol
Anim 2011; 47(2): 132-8.
Kwon K, Hasseman J, Latham S, et al. Recombinant expression
and functional analysis of proteases from Streptococcus pneumo-
niae, Bacillus anthracis, and Yersinia pestis. BMC Biochem 2011;
Walhout AJ, Temple GF, Brasch MA, et al. GATEWAY recombi-
national cloning: application to the cloning of large numbers of
open reading frames or ORFeomes. Methods Enzymol 2000; 328:
Reboul J, Vaglio P, Tzellas N, et al. Open-reading-frame sequence
tags (OSTs) support the existence of at least 17,300 genes in C. el-
egans. Nat Genet 2001; 27(3): 332-6.
Haun RS, Moss J. Ligation-independent cloning of glutathione S-
transferase fusion genes for expression in Escherichia coli. Gene
1992; 112(1): 37-43.
Aslanidis C, de Jong PJ. Ligation-independent cloning of PCR
products (LIC-PCR). Nucleic Acids Res 1990; 18(20): 6069-74.
Ohana RF, Hurst R, Vidugiriene J, Slater MR, Wood KV, Urh M.
HaloTag-based purification of functional human kinases from
mammalian cells. Protein Expr Purif 2011; 76(2): 154-64.
Los GV, Wood K. The HaloTag: a novel technology for cell imag-
ing and protein analysis. Methods Mol Biol 2007; 356: 195-208.
Janssen DB. Evolving haloalkane dehalogenases. Curr Opin Chem
Biol 2004; 8(2): 150-9.
Ramachandran N, Hainsworth E, Bhullar B, et al. Self-assembling
protein microarrays. Science 2004; 305(5680): 86-90.
Kato K, Sato H, Iwata H. Immobilization of histidine-tagged re-
combinant proteins onto micropatterned surfaces for cell-based
functional assays. Langmuir 2005; 21(16): 7071-5.
Braun P, Hu Y, Shen B, et al. Proteome-scale purification of hu-
man proteins from bacteria. Proc Natl Acad Sci USA 2002; 99(5):
Terpe K. Overview of tag protein fusions: from molecular and
biochemical fundamentals to commercial systems. Appl Microbiol
Biotechnol 2003; 60(5): 523-33.
MacBeath G, Schreiber SL. Printing proteins as microarrays for
high-throughput function determination. Science 2000; 289(5485):
Labrou NE. Design and selection of ligands for affinity chromatog-
raphy. J Chromatogr B Analyt Technol Biomed Life Sci 2003;
Urh M, Simpson D, Zhao K. Affinity chromatography: general
methods. Methods Enzymol 2009; 463: 417-38.
Urh M, Hartzell D, Mendez J, Klaubert DH, Wood K. Methods for
detection of protein-protein and protein-DNA interactions using
HaloTag. Methods Mol Biol 2008; 421: 191-209.
Van Reeth T, Dreze PL, Szpirer J, Szpirer C, Gabant P. Positive
selection vectors to generate fused genes for the expression of his-
tagged proteins. Biotechniques 1998; 25(5): 898-904.
Stols L, Gu M, Dieckman L, Raffen R, Collart FR, Donnelly MI. A
new vector for high-throughput, ligation-independent cloning en-
coding a tobacco etch virus protease cleavage site. Protein Expr Pu-
rif 2002; 25(1): 8-15.
Shih YP, Kung WM, Chen JC, Yeh CH, Wang AH, Wang TF.
High-throughput screening of soluble recombinant proteins. Protein
Sci 2002; 11(7): 1714-9.
Hammarstrom M, Hellgren N, van Den Berg S, Berglund H, Hard
T. Rapid screening for improved solubility of small human proteins
produced as fusion proteins in Escherichia coli. Protein Sci 2002;
 Nishihara K, Kanemori M, Kitagawa M, Yanagi H, Yura T. Chap-
erone coexpression plasmids: differential and synergistic roles of
DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an al-
lergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl
Environ Microbiol 1998; 64(5): 1694-9.
Bruckner A, Polge C, Lentze N, Auerbach D, Schlattner U. Yeast
two-hybrid, a powerful tool for systems biology. Int J Mol Sci
2009; 10(6): 2763-88.
Nath N, Hurst R, Hook B, et al. Improving protein array perform-
ance: focus on washing and storage conditions. J Proteome Res
2008; 7(10): 4475-82.
Kubori T, Matsushima Y, Nakamura D, et al. Supramolecular
structure of the Salmonella typhimurium type III protein secretion
system. Science 1998; 280(5363): 602-5.
Cornelis GR, Wolf-Watz H. The Yersinia Yop virulon: a bacterial
system for subverting eukaryotic cells. Mol Microbiol 1997; 23(5):
Galan JE, Collmer A. Type III secretion machines: bacterial de-
vices for protein delivery into host cells. Science 1999; 284(5418):
Diepold A, Amstutz M, Abel S, Sorg I, Jenal U, Cornelis GR.
Deciphering the assembly of the Yersinia type III secretion injecti-
some. EMBO J 2010; 29(11): 1928-40.
Galan JE, Wolf-Watz H. Protein delivery into eukaryotic cells by
type III secretion machines. Nature 2006; 444(7119): 567-73.
Kwon K, Grose C, Pieper R, Pandya GA, Fleischmann RD, Peter-
son SN. High quality protein microarray using in situ protein puri-
fication. BMC Biotechnol 2009; 9: 72.
Nilles ML, Williams AW, Skrzypek E, Straley SC. Yersinia pestis
LcrV forms a stable complex with LcrG and may have a secretion-
related regulatory role in the low-Ca2+ response. J Bacteriol 1997;
Sarker MR, Neyt C, Stainier I, Cornelis GR. The Yersinia Yop
virulon: LcrV is required for extrusion of the translocators YopB
and YopD. J Bacteriol 1998; 180(5): 1207-14.
Matson JS, Nilles ML. LcrG-LcrV interaction is required for con-
trol of Yops secretion in Yersinia pestis. J Bacteriol 2001; 183(17):
Lawton DG, Longstaff C, Wallace BA, et al. Interactions of the
type III secretion pathway proteins LcrV and LcrG from Yersinia
pestis are mediated by coiled-coil domains. J Biol Chem 2002;
Matson JS, Nilles ML. Interaction of the Yersinia pestis type III
regulatory proteins LcrG and LcrV occurs at a hydrophobic inter-
face. BMC Microbiol 2002; 2: 16.
Arifuzzaman M, Maeda M, Itoh A, et al. Large-scale identification
of protein-protein interaction of Escherichia coli K-12. Genome
Res 2006; 16(5): 686-91.
Butland G, Peregrin-Alvarez JM, Li J, et al. Interaction network
containing conserved and essential protein complexes in Es-
cherichia coli. Nature 2005; 433(7025): 531-7.
Kainz M, Gourse RL. The C-terminal domain of the alpha subunit
of Escherichia coli RNA polymerase is required for efficient rho-
dependent transcription termination. J Mol Biol 1998; 284(5):
Schauer AT, Cheng SW, Zheng C, et al. The alpha subunit of RNA
polymerase and transcription antitermination. Mol Microbiol 1996;
Zhang G, Darst SA. Structure of the Escherichia coli RNA poly-
merase alpha subunit amino-terminal domain. Science 1998;
Mah TF, Kuznedelov K, Mushegian A, Severinov K, Greenblatt J.
The alpha subunit of E. coli RNA polymerase activates RNA bind-
ing by NusA. Genes Dev 2000; 14(20): 2664-75.
Mencia M, Monsalve M, Rojo F, Salas M. Substitution of the C-
terminal domain of the Escherichia coli RNA polymerase alpha
subunit by that from Bacillus subtilis makes the enzyme responsive
to a Bacillus subtilis transcriptional activator. J Mol Biol 1998;
Kuo MH, Allis CD. In vivo cross-linking and immunoprecipitation
for studying dynamic Protein:DNA associations in a chromatin en-
vironment. Methods 1999; 19(3): 425-33.
Solomon MJ, Larsen PL, Varshavsky A. Mapping protein-DNA
interactions in vivo with formaldehyde: evidence that histone H4 is
retained on a highly transcribed gene. Cell 1988; 53(6): 937-47.
Improving Soluble Expression and Applications in Protein Functional Analysis Current Chemical Genomics, 2012, Volume 6 17 Download full-text
 Odom DT, Zizlsperger N, Gordon DB, et al. Control of pancreas
and liver gene expression by HNF transcription factors. Science
2004; 303(5662): 1378-81.
Buck MJ, Lieb JD. ChIP-chip: considerations for the design, analy-
sis, and application of genome-wide chromatin immunoprecipita-
tion experiments. Genomics 2004; 83(3): 349-60.
Horak CE, Mahajan MC, Luscombe NM, Gerstein M, Weissman
SM, Snyder M. GATA-1 binding sites mapped in the beta-globin
locus by using mammalian chIp-chip analysis. Proc Natl Acad Sci
USA 2002; 99(5): 2924-9.
Ren B, Robert F, Wyrick JJ, et al. Genome-wide location and func-
tion of DNA binding proteins. Science 2000; 290(5500): 2306-9.
Weinmann AS, Farnham PJ. Identification of unknown target genes
of human transcription factors using chromatin immunoprecipita-
tion. Methods 2002; 26(1): 37-47.
 Euskirchen GM, Rozowsky JS, Wei CL, et al. Mapping of tran-
scription factor binding regions in mammalian cells by ChIP: com-
parison of array- and sequencing-based technologies. Genome Res
2007; 17(6): 898-909.
Impey S, McCorkle SR, Cha-Molstad H, et al. Defining the CREB
regulon: a genome-wide analysis of transcription factor regulatory
regions. Cell 2004; 119(7): 1041-54.
Wei CL, Wu Q, Vega VB, et al. A global map of p53 transcription-
factor binding sites in the human genome. Cell 2006; 124(1): 207-
Toth J, Biggin MD. The specificity of protein-DNA crosslinking by
formaldehyde: in vitro and in drosophila embryos. Nucleic Acids
Res 2000; 28(2): e4.
Hartzell DD, Trinklein ND, Mendez J, et al. A functional analysis
of the CREB signaling pathway using HaloCHIP-chip and high
throughput reporter assays. BMC Genomics 2009; 10: 497.
Received: May 17, 2011
© Peterson and Kwon; Licensee Bentham Open.
This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licen-
ses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.
Revised: July 13, 2012 Accepted: July 18, 2012