Review TRENDS in Biotechnology
10 October 2004
Hydrophobic peptide tags as tools in
bioseparation
Sara Fexby1 and Leif Bülow2
1
Present address: Laboratoire de Technologie Enzymatique, UMR 6022 du CNRS, Université de Technologie de Compiègne,
B.P. 20529, F-60205 Compiègne Cedex, France
2
Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund,
Sweden
Hydrophobic interactions are highly selective, and
differences in surface hydrophobicities between pro-
teins can be used as an efficient handle to facilitate
protein isolation. Aromatic amino acid residues are of
particular importance for molecular recognition because
they have a key role in several biological functions. The
hydrophobicity of a protein can easily be altered with
minor genetic modifications, such as site-directed
mutagenesis or fusions of hydrophobic peptide tags.
An important advantage of hydrophobic peptide tags
over traditional affinity tags is the possibility of exploring
simple and inexpensive bioseparation materials. Recent
results demonstrate the potential of hydrophobic inter-
action chromatography and aqueous two-phase sys-
tems as tools to study relative hydrophobicities of
recombinant proteins with only minor alterations.
This review focuses on hydrophobic peptide tags as
fusion partners, which can be used as important
tools in bioseparation.
Hydrophobic interactions are one of the major driving
forces behind the folding of peptides and proteins. In
addition, hydrophobic interactions are highly selective
and are involved in several biological events, such as
regulation and transport across membranes, enzyme
catalysis, association of protein subunits and protein
aggregation. Lo Conte et al. studied 75 different protein–
protein complexes. The interfaces in the complexes were
much richer in aromatic residues (tyrosine, tryptophan,
phenylalanine and histidine) than the protein surfaces
that remained accessible to the solvent [1]. Hydrophobic
residues are more deeply buried in protein structures than
are hydrophilic residues and, therefore, introduction of
surface-exposed hydrophobic regions in the target protein
can facilitate protein isolation and purification. The
aromatic amino acid residues are highly selective and
important for protein recognition in several biological
functions. For instance, the aromatic residues phenyl-
alanine and tyrosine have key roles in protein–RNA
binding [2]; furthermore, tyrosine residues contribute to
one-sixth of the antigen–antibody interface [1].
The importance of developing techniques for the
isolation and purification of proteins must not be
Corresponding author: Sara Fexby (sarafexby@yahoo.se).
www.sciencedirect.com 0167-7799/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.08.005
Vol.22 No.
underestimated. Several different methods have been
used for bioseparation, all of which exploit differences in
physical, chemical and/or functional properties of the
proteins. To obtain a high degree of purity of the target
protein, several different purification steps are often
needed. Given that the cost for the bioseparation processes
increases with the number of purification steps used, as
well as with consumed time for the processes.
Separation of biomolecules in hydrophobic interaction
chromatography (HIC) occurs on the basis of hydrophobic
interactions between immobilised hydrophobic ligands
and hydrophobic solvent-exposed regions (patches) on
biomolecules. A protein frequently has hydrophobic
patches on its surface and when these are in contact
with an aqueous solvent, the water molecules close to the
hydrophobic patches are arranged in an ordered mode
(Figure 1). This creates a thermodynamically unfavour-
able situation because it represents a decrease in entropy
by comparison with a non-solvated protein plus free water
molecules. The displacement of ordered water molecules
surrounding hydrophobic ligands and proteins leads to an
increase in entropy, resulting in a negative change in free
energy (DG), according to the Gibbs function (DGZDHK
TDS, where DH and DS are the changes in enthalpy and
entropy, respectively). Under normal conditions, the
adsorption enthalpy is small and this indicates that
adsorption is mainly an entropy-driven process [3,4].
Another bioseparation technique, by which hydro-
phobic interactions can be explored, is the aqueous two-
phase system (ATPS). ATPSs are particularly useful for
primary recovery of biomolecules, and as tools for studying
surface properties. Two-phase systems are formed when
an aqueous solution of two structurally different polymers,
or a polymer and salt, separate into two phases when the
concentrations of the components are higher than a
critical threshold value. Each phase is enriched in one of
the phase-forming components and the partitioning of a
protein depends on the characteristics of the protein and
the properties of the two-phase system [5]. The technique
constitutes a mild method of separation of biomolecules as
a result of high water content in both phases (70–95%).
Traditionally, HIC and ATPS have been used for
protein separation of cellular extracts; however, these
techniques can be used as tools for measuring relative
hydrophobicities [6–8]. HIC retention is the result of
512 Review TRENDS in Biotechnology Vol.22 No.10 October 2004

W
WW W
W WWW WW
W W W W
L W + W
L + W
W W W W W
WWW W WW WW W W
WW W
WW W

TRENDS in Biotechnology

Figure 1. Water molecules (W) are highly ordered around hydrophobic solvent-exposed regions, such as hydrophobic ligands (L) and hydrophobic solvent-exposed protein
patches. The specific ligand–protein interaction is thermodynamically favourable because it results in a release of the ordered water molecules.

specific protein–surface interactions, whereas ATPSs according to their hydrophobic properties. The residues
reflect overall net surface properties such as hydrophobi- are given a relative hydrophobicity value, which depends
city. Here, we describe the possibility of using hydrophobic on the method chosen to determine the hydrophobic
peptide tags as tools in bioseparation and discuss the interactions. These interactions are most commonly
possible evaluation of differences in relative hydrophobi- studied by partitioning of amino acid residues, peptides
cities using HIC and ATPS techniques. or proteins between an oil phase and a water phase [6].
Hydrophobicity scales can also be based on peptide
retention times in chromatographic techniques [6] or
Design of hydrophobic peptide tags partitioning of peptides in ATPSs [22,23]. A general
A selective molecular recognition site can genetically be trend in ATPS is that the aromatic residues partition to
introduced on a target protein to facilitate its purification the more hydrophobic top phases, such as poly(ethylene
and isolation. Several so-called ‘affinity tags’ are available, glycol) (PEG) or copolymers of ethylene oxide-propylene
ranging from short peptide sequences to fusion partners of oxide (EOPO), whereas the charged residues move to the
protein size and complexity [9]. Polyhistidine tags are hydrophilic bottom phase.
commonly used for bioseparation, wherein the interaction Proline residues have a rigid ring structure and
between histidine residues and divalent metal ions, such prevent secondary structure formation. Adding prolines
as nickel, copper, zinc or cobalt, are utilized [10]. Bio- between a hydrophobic tag and the target protein could
separation can also be performed via thermosensitive have a positive effect because the degree of surface
elastine tags; a temperature increase results in precipi- exposure of the tag might increase. Variants of N- and
tation of the tagged protein [11]. Target proteins fused with C-terminal hydrophobic peptide tags, fused to different
short hydrophobic peptide tags have successfully been used target proteins, have been evaluated in ATPSs [15,20]. All
in bioseparation (Table 1) because additional hydrophobic exhibited improved hydrophobic properties when proline
residues have a strong effect on the relative hydrophobi- residues were added, as a result of the higher degree of
city of the tagged protein [12–15]. The most commonly surface exposure of the aromatic residues. However, the
used are tryptophan-containing tags [12–14,16,17]; how- difference in solvent exposure between similar combin-
ever, other hydrophobic peptide tags have been examined, ations, such as Y3P2- and (YP)3-tags fused to a mutated
including polyphenylalanines [18], polyisoleucines [19] variant of green fluorescent protein, GFPuv, was minor
and polytyrosines [15,20,21]. [21]. The fluorescence emission for tryptophan residues
When designing a hydrophobic fusion tag, the contri- depends on the polarity of the medium around the
bution of both the individual residues and their combined residues, and this can be used to investigate their degree
effects are of significance. Several hydrophobicity scales of exposure. Tryptophan-containing tags fused to cutinase
are available within which amino acids are ranked from Fusarium solari pisi proved to be solvent-exposed,
Table 1. Hydrophobic peptide tags that have been explored for because the tryptophans in the tags exhibited similar
protein purification emission spectra as free tryptophan peptides [24].
Tag Target protein Refs
(TrpPro)2 Cutinase [14] Applications in bioseparation
ZZ-cutinase [16] Partitioning in ATPS
Endoglucanase I [17] When specific properties, such as the hydrophobicity of a
(TrpPro)4 Cutinase [14]
ZZ-cutinase [16]
protein, are to be evaluated, minimised charge-dependent
Endoglucanase I [17] salt effects are desirable. The charge effects can be reduced
(Tyr)3 Lactate dehydrogenase [15] if partitioning occurs at a pH level that is equal to the
Green fluorescent protein [21] isoelectric point of the protein, or at a point when the
(TyrPro)3 Green fluorescent protein [21]
anions and cations are evenly distributed over the two
(Tyr)3(Pro)2 Lactate dehydrogenase [15]
Green fluorescent protein [21] phases in the system. ATPSs that contain potassium
(Tyr)4 ZZ-Cutinase [20] sulphate (K2SO4) as the dominating salt are commonly
(TyrPro)4 ZZ-Cutinase [20] used for hydrophobicity studies, because K2SO4 gives
(Tyr)6 Lactate dehydrogenase [15] potential differences close to zero in several ATPSs [16,25].
(Tyr)6(Pro)2 Lactate dehydrogenase [15]
Fusions of tryptophan-containing tags to cutinase
(Tyr)8 ZZ-Cutinase [20]
enhanced partitioning in a PEG/salt system to the more
www.sciencedirect.com
 Review TRENDS in Biotechnology
hydrophobic PEG-rich phase, with an increasing number
of tryptophan residues [13]. In another study, tags
containing tryptophan or isoleucine residues were com-
pared, and the aromatic tryptophan residues improved
the partitioning of the tagged protein to the more hydro-
phobic top phase to a higher extent that the isoleucine
residues did [19]. The aromatic tryptophan residues
improved the partitioning of the tagged protein to the
more hydrophobic top phase to a higher extent than it did
the isoleucine residues. Similarly, increasing the number
of tyrosine residues fused to lactate dehydrogenase, LDH,
enhanced the partitioning of LDH to the more hydro-
phobic top phase [15].
Bandmann et al. screened a large number of random-
ized peptides containing nine amino acid residues via the
partitioning of a phage display library in a PEG/salt
system [20]. Several tyrosine-containing peptides were
present in the more hydrophobic PEG-rich phase. The
other aromatic residues, tryptophan and phenylalanine,
which had earlier been found to partition to a high extent
to hydrophobic phases [13,23,25], were only found in small
amounts in the resulting peptides in the hydrophobic
phase [20]. The significant presence of tyrosine residues
could be due to their more common occurrence on protein
surfaces than tryptophans and phenylalanines [26].
Up to 90% of enzyme activity could be extracted in a
detergent-based ATPS in a single step, from Saccharomyces
cerevisae culture broth expressing tryptophan-tagged cuti-
nase [14]. The copolymer EOPO is thermoresponsive, and
heating the polymer solution above its cloud point will
result in phase separation into a water phase and a
polymer phase. The advantage is that the protein can be
recovered in the water phase and the polymer can be
recycled (Figure 2) [27]. Escherichia coli cell extracts of
native and tyrosine-tagged GFPuv proteins were also
partitioned in an EO30PO70 (30% EO and 70% PO)/dextran
system [21]. The hydrophobic EO30PO70 top phase was
heated and the target protein was obtained in a water
solution. A purification factor of 4.5 and an 83% yield were
observed for Y3P2-GFPuv, compared with a purification
factor of 1.9 and a 46% yield for the native GFPuv. Kepka
et al. also demonstrated a successful scale-up of a
thermoseparating ATPS, in which the target protein
EOPO copolymer
and water
Transfer of top phase
Dextran
and water
Target protein
Contaminating
proteins
Recycling of EOPO copolymer
Figure 2. Recycling of the ethylene oxide–propylene oxide (EOPO) copolymer. The EOPO copolymer is enriched in the top phase in an aqueous two-phase system, containing
EOPO and dextran. The copolymer can be recycled if the top phase is transferred and heated above the cloud point of the EOPO copolymer. The thermoresponsive copolymer
precipitates and the proteins are obtained in the water phase.
www.sciencedirect.com
Vol.22 No.10 October 2004 513
(tryptophan-tagged cutinase in 500 L Escherichia coli
homogenate) was obtained in a water phase after a total
recovery over the extraction steps of 71% and a purifi-
cation factor of 2.5 [28].
Bioseparation using HIC
HIC takes advantage of the hydrophobicity of proteins for
selective interactions between proteins and ligands. The
protein size is relevant because a larger protein has a
higher probability of forming multi-point attachments to
the ligand, resulting in stronger interactions and
increased residence time on the column. However, the
hydrophobic solvent-exposed surfaces are often of greater
importance. Fausnaugh and Regnier investigated HIC
separations of different lysozyme isozymes [29]. Differ-
ences of only a few hydrophilic or charged residues
substantially altered retention times on a phenyl-based
HIC column.
Hassinen et al. investigated the separation of hydro-
phobic fusion tags, containing tryptophan or isoleucine
residues, fused to the ZZ protein (the Z domain is the IgG
interacting domain of protein A) on PEG-based and
propyl-based HIC columns [19]. The more hydrophobic
propyl-based HIC column increased the retention times of
the fusion proteins by comparison with the PEG-based
column. Tryptophan residues had a greater impact on the
retention times than did isoleucine residues. Recombin-
antly expressed native GFPuv and tyrosine-tagged GFPuv
(Y3P2-GFPuv) were also separated on a phenyl-based HIC
column by a descending salt gradient (S. Fexby, unpub-
lished). The retention time for Y3P2-GFPuv was pro-
longed compared to that of native GFPuv.
Limitations of using hydrophobic peptide tags
Fusions with less common amino acid residues, such as
hydrophobic residues, might have a negative impact on
the tagged protein, and might effect both protein
expression and stability. Several investigations of ter-
minal fusions of hydrophobic peptide tags have led to
changes in expression. There are several reasons for this
(see Box 1), for example, hydrophobic fusion can cause the
protein to be membrane-associated [18] or alternatively
proteolytic cleavage can reduce the amount of protein
Heat
Protein
and water
EOPO copolymer
TRENDS in Biotechnology
514 Review TRENDS in Biotechnology
Box 1. Extension of peptide tags might affect expression
levels of target proteins
Addition of peptide tags can affect the expression levels of both
genes and proteins. This is because:
(1) peptide tags influence the mRNA stability
(2) they affect the translation efficiency
(3) rare codons can interfere with ribosome trafficking
(4) the tagged protein is partly/fully insoluble
(5) the tagged protein becomes membrane associated
(6) there is proteolytic degradation of the tagged protein
obtained [17,19]. Collén et al. fused various tryptophan-
containing peptides to endoglucanase I from Trichoderma
reesei, resulting in a decrease in the expression level of all
tagged proteins [17]. Changes in mRNA stability can also
affect the expression levels. N-terminal hydrophobic
fusions, containing three tyrosine and two or three proline
residues fused to GFPuv, favourably influence the
expression of the tagged protein [21]. This is probably
due to changes in mRNA stability, because the amount of
corresponding mRNAs was found to correlate with the
amounts of proteins expressed.
Increased hydrophobic properties might result in
protein association of dimers or larger aggregates, or in
dissociation into smaller subunits [5]. The conformational
changes might lead to denaturation and collapse of the
protein. These irreversible conformational changes are
favoured in the boundary layer between hydrophilic and
hydrophobic environments, such as the liquid–solid inter-
faces of a stationary HIC phase. This can complicate the
use of the separation processes in HIC and ATPSs of
hydrophobic tagged proteins.
Design of novel separation media that are useful for HIC
Depending on the magnitude of the changes in free energy
for favourable protein–ligand interaction, DGassoc, mol-
ecular forces can be generated that exceed the free energy
that is associated with the folding of the protein, DGfold,
resulting in partial or complete denaturation of the
protein [4]. For example, separation of a-lactalbumin on
phenyl-based HIC columns resulted in elution of several
peaks, as the adsorption of a-lactalbumin induced confor-
mational changes of the protein [30]. Furthermore,
separation of an E. coli protein extract containing
tyrosine-tagged LDH (Y6P2-LDH) on phenyl-based HIC
100/0
40/60
Figure 3. A schematic illustration of a novel HIC medium with altered hydrophobic properties. A series of the novel media was obtained by varying the reaction ratios of the
two monomers HBVE (hydroxybutyl vinyl ether) and BE (butyl vinyl ether) in the polymer grafting reactions. The reaction ratio of HBVE (RHBVE) and BVE (RBVE) in each
medium is written as RHBVE/RBVE above each illustration. The hydrophobic properties increased as the reaction ratio of the more hydrophobic monomer, BVE, increased from
0% to 80% in the polymer grafting reaction.
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Vol.22 No.10 October 2004
columns resulted in the recovery of inactive enzyme
(S. Fexby, unpublished). The tyrosine extensions thus
caused stronger protein–ligand interactions than did
native LDH protein, but at the expense of lost enzymatic
activity. This indicates unwanted conformational changes
of the proteins, owing to adsorption. A more hydrophobic
medium might cause too strong a protein–ligand inter-
action, which can result in partial or complete denatura-
tion of the protein; therefore, it is of importance to use
adsorbent media that will not cause serious conformation-
al changes in the target protein. A novel medium has been
synthesized by radical initiated polymer grafting of vinyl
ether reagents at Sepharosee HP base matrices [31]. The
advantage of the novel medium is the afforded possibility
of altering the hydrophobic properties of the medium by
changing only the reaction ratios of the two monomers in
the polymer grafting reaction (Figure 3). Upon investi-
gation, it was found that the hydrophobicity could be
tuned and as the media surface hydrophobicity increased,
so did the protein retention times; the less hydrophobic
medium proved to exhibit the highest selectivity (S. Fexby
et al. unpublished).
Studies of protein surface hydrophobic alterations
Biological functions of many proteins are regulated by
conformational changes. For example, many calcium-
binding proteins expose hydrophobic areas upon binding
of calcium, and these changes could be used for binding to
hydrophobic chromatography matrices in the presence of
calcium [32]. Furthermore, Gulyaeva et al. were able to
differentiate between compounds that are capable of
crossing the blood–brain barrier and those that cannot,
using a combination of measurements from ATPSs and
HPLC [33]. Protein surfaces and surface changes as a
result of mutations can also be studied in ATPSs, because
protein partitioning depends on the surface properties of
the protein. The influence of surface-exposed amino acid
residues on the protein partitioning has been evaluated in
a study by Berggren et al. [7]. Cutinase variants were
studied that differed in one or several surface-exposed
amino acid residues as a result of site-directed muta-
genesis, and conformational changes could be observed. In
a study by Fexby et al., native LDH and seventeen
different tagged LDH proteins were evaluated with HIC
and ATPS [8]. The LDH variants differed only slightly in
80/20 60/40
20/80
HBVE
BVE
TRENDS in Biotechnology
 Review TRENDS in Biotechnology
primary structure, size and pI value, owing to the fusion of
randomized pentapeptides. The order of retention times
for separation on phenyl-based HIC columns correlated
well with the partitioning coefficients that were obtained
from EO30PO70/dextran systems; the correlation coeffi-
cient, R2, was 0.89. The results of this study demonstrate
the sensitivity of both methods to slight variations in
protein primary structure and surface hydrophobicity.
Predictions of relative hydrophobicity
Hydrophobicity scales have been used to predict certain
structural features of proteins in several studies. For
example, Palliser and Parry located b-strands on the
surface of proteins using hydrophobicity scales [34]. More
importantly, antigenic sites and membrane-associated
regions have also been predicted using methods based on
hydrophobicity scales [35,36]. However, when comparing
the numerous published hydrophobicity scales, the vari-
ation in hydrophobicity ranking of individual amino acid
residues is remarkable [6,37]. Differences between the
various scales could be due to several factors. The
amphiphilic character of the aromatic amino acids
(phenylalanine, trytophan and tyrosine) leads to different
relative contributions to the hydrophobicity, depending on
the method and solute chosen [6,37,38]. The assumption
regarding the charged or uncharged state of histidine
residues affects the hydrophobicity ranking [8], and also,
cysteine residues can form disulphide bonds, and the
cysteine residues then appear more hydrophobic [6].
The hydrophobic ranking of these amino acid residues
varies considerably.
Calculated relative hydrophobicity contributions must
be handled carefully and they can only complement
experimental results. For example, N-terminal tagging
of LDH with LeuValGlyValIle or LeuTyrGlyCysIle,
respectively, results in much less hydrophobic proteins
according to HIC and ATPS results, than do theoretical
calculations using hydrophobicity scales [8].
Conclusion
The hydrophobic properties of a target protein can be
altered by tagging with a short hydrophobic peptide, and
these changes can be explored in separation. Tryptophan
residues are more hydrophobic in nature than tyrosines;
despite this, the decrease in hydrophobic properties can be
compensated by increased expression and higher stability
of the target protein. The combined properties of the
residues in the added peptide tag, together with the target
protein, are of importance. Therefore, a single universal
optimal hydrophobic peptide tag does not exist. Addition-
ally, using hydrophobicity scales only for predictions of
relative hydrophobicity of peptide tags is not advisable
because neighbouring residues of the target protein must
be taken into account. The environment surrounding a
specific residue will thus affect its exposure and its
properties. Until the time when better theoretical predic-
tions become available, a combination of experimental and
theoretical methods is preferable to achieve improved and
efficient protein isolation.
www.sciencedirect.com
Vol.22 No.10 October 2004 515
Acknowledgements
Financial support from the Swedish Centre for Bioseparation is gratefully
acknowledged.
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The Health Internetwork Access to Research Initiative (HINARI) of the WHO has launched a new community scheme with the UN Food
and Agriculture Organization.

As part of this enterprise, Elsevier has given 185 journals to Access to Global Online Research in Agriculture (AGORA). More than 100
institutions are now registered for the scheme, which aims to provide developing countries with free access to vital research that will
ultimately help increase crop yields and encourage agricultural self-sufficiency.

According to the Africa University in Zimbabwe, AGORA has been welcomed by both students and staff. ‘It has brought a wealth of
information to our fingertips’ says Vimbai Hungwe. ‘The information made available goes a long way in helping the learning, teaching
and research activities within the University. Given the economic hardships we are going through, it couldn’t have come at a better time.’

For more information visit:

http://www.healthinternetwork.net

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