Article

pubs.acs.org/crystal

Analysis and Control of Protein Crystallization Using Short Peptide

Tags That Change Solubility without Affecting Structure, Thermal
Stability, and Function
Mohammad Monirul Islam,†,∥ Shigeyoshi Nakamura,§ Keiichi Noguchi,‡ Masafumi Yohda,†
Shun-ichi Kidokoro,§ and Yutaka Kuroda*,†
†
Department of Biotechnology and Life Science, and ‡Instrumentation Analysis Center, Tokyo University of Agriculture and
Technology, Tokyo 184-8588, Japan
§
Department of Bioengineering, Nagaoka University of Technology, Niigata 940-2188, Japan
∥
Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong 4331, Bangladesh
*
S Supporting Information

ABSTRACT: Short peptide tags attached to recombinant

proteins are emerging as an important tool for biochemical
research. Here, we report the effects of 10 Solubilization
Controlling Peptide (SCP) tags on crystallization behavior of a
bovine pancreatic trypsin inhibitor (BPTI) variant. The tags
did not affect structure, thermodynamics, and activities of
BPTI. Moreover, eight of the tagged variants crystallized under
the same condition, and six of them diffracted at high
resolution. All variants with long-term solubility (LS) between
1 and 6 mg/mL produced crystals that diffracted well, while
variants with LS < 1 and >6 mg/mL did not crystallize, produced poorly diffracting crystals, or crystallized under a different
condition. The only exception was a glutamine tagged variant, which had an LS of 5 mg/mL, but fast aggregation kinetics, and
produced mere needles unsuitable for further analysis. Crystal structures indicated that most tags were largely invisible, indicating
high flexibility, without having interactions with nearby residues. Therefore, short peptides, introducing a mere 5−7 residue
elongation, could provide a useful technology for tuning protein solubility without affecting its other properties and hence for
overcoming problems associated with excessively low or high solubility, such as in crystallization.

■ INTRODUCTION
Manipulation of protein solubility is becoming a central issue in
several aspects of biotechnological and pharmaceutical usage of
recombinant proteins.1 Empirical attempts to control protein
solubility by amino acid substitution have been reported,2−4 but
the application range of such attempts remains limited as one
cannot yet control protein solubility without affecting other
properties.
Short peptide tags have recently been proposed for
improving protein solubility and purification.5,6 They are
significantly smaller than traditional solubilizing protein tags,
which constitutes a major advantage.7 Furthermore, they are
anticipated to function as unfolded “tails”, independent from
the proteins to which they are attached.7 This makes short
peptide tags particularly attractive as they confer the potential
for manipulating a protein’s solubility without affecting its
structural or biochemical properties. For example, short five-
residue Lys and Arg tags increased the solubility of a bovine
pancreatic trypsin inhibitor (BPTI) variant by over 4-fold
without altering either its NMR spectrum or activity,8 and other
five-residue peptide tags could be used to control protein
solubility in a pH-dependent manner.9
The production of crystals is a prerequisite first step for
determining high resolution structures of biomolecules, but the
biophysical mechanism of crystallization remains to be fully
understood, and the success rate of protein crystallization
projects is surprisingly low, even for well folded proteins with
moderate sizes. Though several factors can affect protein
crystallization, solubility is a critical, and relatively uncharac-
terized one.10−13 A few rationally designed point mutations
intended to improve a protein’s crystallization behavior by
altering its intrinsic properties, such as crystal contacts,
solubility, or local flexibility, have been occasionally re-
ported.14−18 The conclusions were somewhat mixed, especially
those concerning the role of protein solubility as their effects
were entangled with those of other factors.19
Here, we report a systematic investigation of the influence of
short solubility-controlling peptide tags on the structure,
thermodynamics, and crystallization behavior of our model
protein, BPTI-19A, for 10 amino acid types [acidic (D and E),
basic (K and R), polar (S, N, Q, P, and H), and hydrophobic
Received: January 5, 2015
Revised: April 21, 2015
Published: May 8, 2015

© 2015 American Chemical Society 2703 DOI: 10.1021/acs.cgd.5b00010

Cryst. Growth Des. 2015, 15, 2703−2711
Crystal Growth & Design
Figure 1. Effects of the SCP tags on protein structures. Top: Superimposed backbone structures of untagged BPTI-19A (black lines) and the tagged
variants (gray lines). The overall backbone RMS deviations were 0.2−0.3 Å, indicating the retention of the same natively folded structures in all
variants. Bottom: Sequences of the untagged and tagged BPTIs are shown with underlined SCP tag residues. Two Gly residues were added as a
spacer (C2G), followed by five amino acid residues (C5X) of the same type.
(I)]. None of the tags affected the trypsin inhibitory activity,
thermodynamic properties, or structure of BPTI-19A, whereas
the crystallization properties were clearly influenced by the
changes in long-term solubility (LS) and precipitation speed
generated by the tags.19 The results show that short solubility
controlling peptide tags could offer a promising method for
analyzing and controlling the effect of solubility on protein
crystallization, by isolating it from interfering factors.
■
METHODS
Mutant Design, Expression, and Purification. All BPTI
variants were constructed using a pMMHa vector as previously
described.21 DNA sequences corresponding to two Gly residues (as a
spacer) and the SCP tags were added to the C-terminus of the model
BPTI using QuikChange site directed mutagenesis, and constructs
were confirmed by DNA sequencing (Figure 1). All poly amino acid
tagged variants were overexpressed in an Escherichia coli JM109(DE3)-
pLysS cell line and purified by reverse phase HPLC. The protein
identities were confirmed by ESI-TOF mass spectroscopy, and purified
proteins were preserved at −30 °C as lyophilized powder.
DSC Measurement and Data Analysis. Samples for differential
scanning calorimetry (DSC) were prepared by dissolving lyophilized
proteins in 20 mM sodium acetate buffer (pH 4.0, pH4.7 and pH5.5),
followed by extensive dialysis for 18 h at 4 °C as previously
described.20 DSC measurements were performed using a VP-DSC
(Microcal, USA) in the temperature range of 5−80 °C at a scan rate of
1 °C/min (Figure 2; Table 1). The apparent heat capacity curves were
analyzed with a two-state model using a nonlinear least-squares fitting
method and a linear temperature dependence of the heat capacity for
the native and denatured states.21,22
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Circular Dichroism (CD) Measurements. The variant’s thermal
stabilities were monitored using the CD signal at 220 nm measured
using a 1 cm cuvette on a Jasco J-820 spectropolarimeter, as described
earlier.23 Protein samples were prepared at 10 μM concentration in 20
mM acetate (pH 4.7) and 20 mM Tris-HCl (pH8.7) in the presence of
0.0, 0.5, 0.75, 1.0, and 1.5 M ammonium sulfate. The temperature was
raised by 1 °C/min from 5 to 75 °C.
Crystallization. Protein stock solutions with 10−15 mg/mL
concentration were prepared in 15 mM Tris-HCl, pH 7.0. A 1 μL
drop of the stock was mixed with 1 μL of crystallization solution, and
set for hanging drop vapor diffusion at 20 °C as reported earlier for
BPTI-19A.24 Crystals of C5D, C5H, C5S, C5P, C5Q and C5E were
developed under the same condition as for BPTI-19A and by using
initial protein concentrations between 5 and 10 mg/mL in 20−30%
PEG4000, 0.2 M lithium sulfate, and 0.1 M Tris-HCl pH8.5. The C5R
and C5K did not form crystals large enough for diffraction under this
condition nor under any of the 98 Hampton conditions. C3R formed
large crystals under the above crystallization conditions, but at an
initial protein concentration of 20−30 mg/mL. Similar to C5K, C3K
formed only needles, and we eventually crystallized C3K using a
heteroseeding strategy where microseeds of C3R were used. In the
heteroseeding strategy, microseeds were collected from an initial
crystallization drop containing over 20 mg/mL of C3R and seeded
onto a drop containing 5−10 mg/mL of C3K with all other conditions
remaining the same as in the initial C3R drop. Finally, C5N was
crystallized in 4 M sodium formate using the Hampton screening kit at
20 °C using the hanging drop vapor diffusion technique (Figure 3).
Structure Determination. The X-ray diffraction data were
recorded from single crystals using a synchrotron beamline at the
Photon Factory (PF, Tsukuba, Japan). The data were processed with
the HKL2000 program package, using DENZO for the integration and
SCALEPACK for the merging and statistical analysis of the diffraction
intensities.25 The structures of all variants were determined by
DOI: 10.1021/acs.cgd.5b00010
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Crystal Growth & Design
Figure 2. Effects of the SCP tags on protein stability. (a) Thermal
transition at pH4.7 measured by DSC (bold line: BPTI-19A; solid
lines:tagged variants) and (b) thermodynamic parameters (Tm: ■;
ΔHTm: gray bars) at pH 4.7 are shown. First, the individual heat
capacity curves at pH 4.7 were analyzed (Tm and ΔHTm determined)
with a two-state model using a nonlinear least-squares fitting method.
Then the individual heat capacity curves at pH 4.7 were reanalyzed by
global fitting using the average ΔCp obtained from the individual heat
capacity curves at pH 4.0, 4.7, and 5.5.21,22 Finally, the standard
deviations of the thermodynamic parameters were calculated (shown
as error bars). (c) Ammonium sulfate concentration’s dependence of
thermal stability determined by CD at pH 4.7 (□: BPTI-19A; ○:
C5D; △: C5K; and ◇: C5R) and 8.7 (■: BPTI-19A; ●: C5D; ▲:
C5K; and ◆: C5R).
molecular replacement with the program Molrep and refined using
Refmac526 and SHEXL97.27 Structures were validated using Sfcheck28
and visualized using Coot29 and Xfit.30 The coordinates and structure
factors were deposited in the Protein Data Bank under PDB IDs:
3AUB, 3AUC, 3AUD, 3AUE, 3AUG, 3AUH, and 3WNY.
■ RESULTS AND DISCUSSION
Effects of the SCP Tags on Protein Solubility. Our
model protein, BPTI-19A, is a simplified 58-residue protein,
containing 19 alanines covering a large fraction of its molecular
surface. BPTI-19A has therefore a relatively low solubility but is
natively folded,24,31 and the alanines on protein surface are
anticipated to minimize interactions between the host protein
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and the peptide tags. We designed and constructed 10 BPTI-
19A variants tagged at their C-terminus with 10 representative
types of SCP tags (polar: N, Q, and S; hydrophobic: I;
positively charged; R and K; negatively charged: D and E, along
with P and H).9,19 Two Gly residues were added as a spacer
between BPTI and the tags (Figure 1). The variants were
named according to the number and type of the amino acid
added. For example, C5D stands for BPTI-19A with five Asp
attached to its C-terminus (plus two Gly residues as a spacer
between BPTI and the C5D tag). Additionally, we also
prepared a reference variant, C2G, which has only two glycine
residues and no additional residues (Figure 1). The BPTI
variants’ solubility was measured as the maximum protein
concentration of the supernatant in the presence of 1.5 M
ammonium sulfate at pH4.7 and pH7.7.9,19 The C5K and C5N
increased protein solubility over 9-fold at pH 7.7, while the
C5H, C5Q, C5S and C5R increased protein solubility 2.9 to 5
fold.9 At lower pH, the negatively charged C5D and C5E
merely affected protein solubility (1−2 fold solubilization),
while at higher pH they introduced over 5−7 fold
solubilization. The very hydrophobic Ile markedly decreased
BPTI’s solubility,9 and noteworthy Pro, the most soluble amino
acid with a solubility of 1600 g/L,32 barely affected BPTI’s
solubility.
Effects of the SCP Tag on Thermodynamic Properties.
The effects of SCP tags on protein stability were assessed using
DSC at pH 4.0, 4.7, and 5.5 (Figure 2a,b). All of the variants
exhibited a reversible two-state thermal folding-unfolding
process, except C5D that aggregated at pH5.5. The melting
temperatures (Tm) of the poly amino acid tagged variants were
very similar to that of the C2G variant (±0.5−1 °C), which was
<3 °C lower than that of the untagged BPTI-19A (Figure 2b).
These results suggested that the small stability decrease of the
tagged variants was associated with the addition of the two-Gly
residue spacer rather than to the five amino acid residue tags
(Figure 2b).
None of the SCP tags significantly affected the stability of the
BPTI-19A both in the absence (Figure 2c) and presence of
ammonium sulfate (Supplementary Figure S1, Supporting
Information).9 The melting temperature (Tm) as well as the
calorimetric enthalpy changes at Tm (ΔHTm) were both
essentially unaffected by the addition of five-residue tags
(Figure 2b). In particular, the enthalpy changes of C2G, C5D,
C5H, C5N, C5P, C5Q, C5R, and C5S were a mere 1−3 kcal/
mol lower than that of the untagged BPTI, whereas that of the
C5K decreased by 4.5 kcal/mol (Table 1). This clearly suggests
that all variants remained natively folded under both the
crystallization and solubility measurement conditions. Further-
more, none of the tags decreased the trypsin inhibition activity
of BPTI-19A (Table 1), indicating that the tags act as
independent entities from BPTI-19A to which they are
attached, from both thermodynamic and functional viewpoints.
Finally, although we did not analyze the tag’s influence on the
thermodynamic of crystallization, such insights could help
designing tags tailored to individual proteins and are worth
being investigated in future studies.
Effects of the SCP Tag on Crystallization. Here, we
report observations we made while crystallizing the tagged
variants, because SCP tags offer a unique tool for controlling
protein solubility without affecting its other properties, which
may yield new insights into the effect of protein solubility on
crystallization.17 Most tags merely affected the crystallization
DOI: 10.1021/acs.cgd.5b00010
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Table 1. Solubility, Stability, and Activity of the SCP Tagged BPTI Variants
thermal stabilitya solubilization effectsb effect on crystallizationf
mutants Tm (°C) ΔH (kcal/mol) c
TS (mg/mL) d
LS (mg/mL) e
precipitation speed (mg/mL/h) /(%/h) crystal type structure activityg (%)
BPTI-19A 51.95 60.18 ± 0.74 1.85 ± 0.14 0.74 ± 0.03 0.023/1.29 rod solved 100
C2G* 49.38 58.56 ± 1.49 2.20 ± 0.08 0.80 ± 0.02 0.029/1.32 N.D.i N.D.i 100
C5H 48.57 57.87 ± 1.19 0.86 ± 0.04 0.27 ± 0.01 0.012/1.42 rod solved 100
C5P 48.09 58.98 ± 1.76 1.29 ± 0.09 0.64 ± 0.14 0.014/1.04 rod/plate solved 100
C5S* 48.80 58.72 ± 2.76 4.40 ± 0.19 1.36 ± 0.27 0.063/1.43 rod solved 100
C3R* N.D, N.D. 6.04 ± 0.30 3.30 ± 0.50 0.057/0.94 cubic solved 100
C3K* N.D. N.D. 12.1 ± 0.45 12.1 ± 0.65 ∼0.0/0.00 cubic solved 100
C5D 46.04 60.04 ± 1.36 14.51 ± 0.5 3.15 ± 0.10 0.237/1.63 rod/plate not solved 100
C5E* 47.58 53.03 ± 0.46 14.3 ± 0.51 3.08 ± 0.50 0.131/1.63 rod/plate not solved 100
C5R 48.84 57.49 ± 1.55 9.42 ± 0.25 3.82 ± 0.61 0.117/1.23 needle not solved 100
C5K 49.13 54.84 ± 1.68 >25.00 >25.00 ∼0.0/0.00 needle not solved 100
C5Q 48.19 56.09 ± 1.60 5.15 ± 0.25 1.09 ± 0.07 0.08/1.64 needle not solved 100
C5N 49.67 58.10 ± 0.51 17.66 ± 12.14 ± 0.69 0.115/0.65 hexagonal solved 100
C5Ih 48.88 58.78 ± 0.31 ∼0.0 ∼0.0 −/N.D. no crystal not solved 100
a
Thermodynamic parameters determined by DSC. N.D. stands for not determined. bProtein solubility defined as the protein concentration in the
supernatant of supersaturated protein solution measured in the presence of 1.5 M ammonium sulfate.9,19 To ensure reliability and reproducibility of
the solubility parameters, all solubility values were measured at least three times, in different working days and with different sets of reagent. The
experimental errors (standard deviations) were determined using more than three independent solubility measurements. cTS stands for transient
solubility, and it is determined as the amount of protein remaining in solution after 20 min incubation. dLS stands for long-term solubility, and it is
determined as the amount of protein remained in solution after 48 h incubation at pH 7.7, as described previously.19 Solubility parameters
determined and/or reconfirmed in this study are marked by asterisks. The unmarked values are adapted from ref 19. eThe fraction of protein
precipitated per hour of incubation at pH 7.7 calculated from 48 h incubation values. fEffect of the SCP tags on crystallization of BPTI-19A to which
they were attached. gTrypsin inhibitory activity measured by monitoring the hydrolysis of N-benzoyl-D,L-arginine-p-nitroanilide at equimolar
concentrations of BPTIs and trypsin.40 hC5I precipitated almost completely under the solubility measurement conditions. iCrystallization and
structure determination were not attempted for the C2G variant.

Figure 3. Pictures of the crystals. The variant identities are mentioned on the top of each panel. Crystals of untagged BPTI-19A and its tagged
variants, except for C5N, were all developed using the hanging drop technique at 20 °C, 20−30% PEG-4000, 0.2 M lithium sulfate, 0.1 M Tris-HCl,
pH8.5. Crystals of C5N were grown under the same conditions but with the addition of 4 M sodium formate. In all cases crystals developed within
24−36 h using a hanging drop setting.

2706 DOI: 10.1021/acs.cgd.5b00010

Cryst. Growth Des. 2015, 15, 2703−2711
Crystal Growth & Design
properties per se, and we could crystallize the untagged BPTI-
19A and its C5D, C5H, C5S, C5P, C5Q, and C5E variants
under the same crystallization conditions. Namely, the crystals
were grown at 20 °C using the hanging drop vapor diffusion
technique under 20−30% PEG4000, 0.2 M lithium sulfate, and
0.1 M Tris-HCl, pH 8.5 at an initial protein concentration of
5−10 mg/mL. The C5N crystals were developed under the
same conditions but in the presence of 4.0 M sodium formate
(Figure 3). We thus speculate that excessive solubility might
render the protein difficult to crystallize, and the high sodium
formate concentration could have promoted the crystallization
of C5N by decreasing its high solubility.
On the other hand, we did not obtain crystals of reasonable
sizes for the highly soluble C5R and C5K variants, which
formed small needles, even by raising the initial protein
concentrations to 40−50 mg/mL or using 4.0 M sodium
formate, which was successful for C5N. We thus produced
crystals of the less soluble C3R and C3K variants, which had
3.3 and 6.5 fold solubilization (Table 1) and crystallized more
easily than the respective five-residue tagged variants. The C3R
formed large crystals when the initial concentration was raised
to 20−30 mg/mL, whereas C3K formed needles similar to
C5K. Eventually, we developed large C3K crystals using a
heteroseeding strategy where microseeds from the C3R variants
were used as seeds for the C3K variant. The difficulties in
getting the large crystals of the C5R and C5K variants could
originate from their extreme solubility, but also from strong
local repulsive forces arising from their positively charged tags.
The C5I was incompatible for crystallization, as precipitation
readily appeared in the crystallization drop, under most
conditions even at very low initial protein concentration.
Similar observations were also made with the C3I, while the
C1I remained almost the same as those observed in the case of
reference BPTI-19A.
For the purpose of discussion, we compared the above
crystallization properties with the variant’s transient solubility
(TS) as well as ong term solubility (LS), where TS is the
protein solubility measured after a 20 min centrifugation, which
is a condition often used for preparing crystallization stock
solutions, and LS is the protein’s concentration in the
supernatant of a saturated protein solution measured after 48
h of incubation.20 Mutants that did not crystallize or yielded
poor crystals were located in a TS/LS area where TS is high and
LS low (Figure 4a). Similarly, mutants that produced well
behaved crystals were located around a dotted line drawn in the
TS/LS diagram. All crystals were produced under a similar
condition but for C3K which was crystallized using a
heteroseeding strategy and C5N which was crystallized in the
presence of sodium formate. The crystallization behaviors
correlated well with TS and LS measured at pH7.7 (Figure 4a),
but not with TS and LS measured at pH 4.7 (Figure 4b;
Supplementary Figure S2, Supporting Information) probably
because the crystallization pH was 8.5.
Finally, we discuss the crystallization behaviors with regard to
the precipitation speed and the ratio of TS to LS. Variants with
a slow precipitation of <0.08 mg/mL per hour and that retained
60−70% of proteins in solution after 48 h of incubation yielded
good quality crystals, and their structures could be solved
(Table 1; Figure 4b). However, proteins with high precipitation
speeds were difficult to crystallize, and failure to crystallize C5Q
despite its solubility being within the suitable range, might thus
be related to a high precipitation speed exceeding 0.08 mg/mL
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Figure 4. Effects of SCP tags on protein solubility and crystallization
behavior of the BPTI-19A. The crystallization properties of the tagged
BPTI-19A variants are displayed on a correlation plot between the
long-term solubility (LS) versus transient solubility (TS) at pH 7.7 (a)
and pH 4.7 (b). The graph is displayed with a log2 scale for both axes.
The crystallization behavior is indicated as follows: □: good crystals
and structures solved; ⧫: large crystals but poor diffraction; ▲: needles
(no structure solved). The variants are indicated by the amino acids
contained in their tag; −19 is the reference protein BPTI-19A; C3R
and C3K stand for three residues Arg and Lys tagged BPTI variants,
respectively. Asn (gray box) crystallized under a high salt
concentration condition where its solubility is reduced. The shaded
area in panel (a) indicates the LS/TS region where variants had poor
crystallization properties, and the dotted line indicates an LS/TS curve
around which BPTI-19A variants yielded high resolution crystals.
per hour. Altogether, these observations indicate that both
excessively high and low solubility inhibit proper crystallization.
Structure of SCP Tagged Variants. We determined the
crystal structures of the untagged BPTI-19A and several of its
tagged variants (C3R, C3K, C5S, C5N, C5H, C3E and C5P) at
atomic resolutions (1.0 to 2.3 Å), almost under the same
crystallization conditions (Table 2). The R-factor of the
untagged variant was the lowest, but C5P and C5N yielded
R-factors almost as good as the untagged variant (Table 2).
Although, some tags had a small negative impact on crystal
quality in terms of resolution or R-factor, which might arise
from increased local flexibility at the BPTI-19A termini, most
variants formed crystals that diffracted with resolution better
than 2.3 Å and with R-factors better than 25% (Table 2). The
exceptions were C5D and C5E tagged variants, which readily
crystallized, but did not diffract well even when developed
under different crystallization conditions.
Though the peptide tags did not affect the overall structure
or the physicochemical properties of the model BPTI-19A, they
did change the crystal packing, space group and monomer
orientation in the asymmetric unit (Table 2; Figure. 5). BPTI-
19A, C5P, and C5S had a C2 symmetry and contained the
same number of monomers in the same orientation (Figure
DOI: 10.1021/acs.cgd.5b00010
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Table 2. Crystallization, Refinement, and Structure Determination

C5D/
parameters BPTI-19A C5H C3K C5N C5P C3R C5Ea C5S C5Ib
mol weight (kDa) 5.979 6.779 6.478 6.664 6.579 6.562 6.669/ 6.529 6.659
6.739
space group C2 P21 P21 P6522 C2 P21 not solved C2 not
solved
unit cell parameters a = 53.33 a = 39.01 a = 54.87 a = 61.49 a = 64.31 a = 38.90 a = 64.26
b = 39.06 b = 120.33 b = 71.44 b = 61.49 b = 48.51 b = 71.39 b = 48.39
c = 49.23 c = 53.32 c = 57.24 c = 171.91 c = 36.81 c = 40.33 c = 36.48
α/γ = 90.00 α/γ = 90.00 α/γ = 90.00 α/γ = 90.00 α/γ = 90.00 α/γ = 90.00 α/γ = 90.00
β = 101.59 β = 106.55 β = 92.09 β = 90.00 β = 108.74 β = 92.46 β = 108.89
Matthews coefficient 2.15 2.27 2.16 2.21 2.09 2.19 2.01
solvent content (%) 43.5. 45.9 43.0 43.2 41.2 43.7 38.3
reflection used 49171 15499 79411 14213 19826 65099 7463
max resolution (Å) 1.00 2.28 1.40 1.94 1.40 1.20 1.91
R-factor/R-free 0.14/0.16 0.18/0.25 0.25/0.29 0.17/0.21 0.16/0.18 0.18/0.19 0.19/.026
Ramachandran plot statistics:
most favored region 90.4 85.5 89.6 89.9 89.2 87.7 90.2
(%)
backbone RMSD (Å) 0.307 0.317 0.270 0.270 0.259 0.274
C-termini visibility 58th 64th 56th 57th 59th 57th 58th
Crystal Packing:
Nb monomer/cell 2 8 8 3 2 4 2
rel orientationc A B C D A E A
a
C5D and C5E crystallized readily, but both diffracted poorly, even when crystallized under different crystallization conditions. bBecause of
precipitation, C5I did not crystallize even upon lowering the protein concentration of the stock solution. cRelative orientations of the first chain with
respect to the others in the asymmetric unit as estimated from Figure 5 are classified into 5 classes (A−E). Only mutants that crystallized with the C2
symmetry had the same orientation.

5a−c; Table 2). On the other hand, C3R, C5H and C3K
crystallized with a P21 symmetry, with four chains per unit cells
for C3R, whereas C5H and C3K had eight chains per unit cells
(Figure 5f; Table 2) but with different relative chain
orientation. Finally, the poor diffraction of the C5D and C5E
variants, despite short-term solubility lying in the well behaved
solubility limit, possibly originate from their fast precipitation
speed at pH7.7 (Figure 4; Table 1), resulting in poorly packed
crystals.
The high resolution X-ray structures provided a reliable and
direct assessment of the tag structures and their minimal effects
on the native BPTI-19A structures (Table 2). In all of the poly
amino acid tagged variants, both the backbone and side-chain
structures of BPTI-19A were almost perfectly retained (from
residues 1−56), with average backbone deviation of <0.3 Å.
Noteworthy, the SCP tags did not influence the structure of the
C-termini as the Cα-RMSD of residues 50−56 with respect to
BPTI-19A was 0.201 ± 0.02 Å (the last two terminal residues
[57th and 58th] were not included in the calculation as they are
highly flexible even in the wild-type structures). Finally, most
tags were not visible, as one would expect from highly flexible
regions (Table 2; Figure 5). The only exception was the five-
residue-His tag, which was partially visible (Supplementary
Figure S3, Supporting Information). The tag was fully extended
and pointing away from the BPTI-19A molecular surface. None
of the His residues interacted with the BPTI’s surface residues
or with nearby tag residues (Figure 5h) indicating that the tag
behaves as a structure-context-independent entity. One may
thus assume that, in solution, the SCP tags act as an unfolded
tail and increase protein solubility without introducing any
novel intra or intermolecular interactions. This was further
corroborated by a full trypsin inhibition activity of all of the
tagged variants (Table 1) demonstrating that the tags did not
2708
interact with the BPTI-19A’s binding site of trypsin, in solution
as well as in the crystal structure.
To date, our study investigates tags that behave independ-
ently from the target protein because we believe that the
independency will ensure solubility to be modulated alone.
However, we may speculate that tags tailored to interact with
the target protein could stabilize a marginally stable protein
thus improving its crystallization behavior, as well. Such an
avenue would be worth being investigated in future studies.
Controlling Protein Solubility with Short Peptide
Tags. Several reports demonstrated that the solubility of a
recombinant target protein can be increased by fusing highly
soluble proteins.33−35 However, fusion proteins are large and
have inherent tendencies to perturb the structure, function, and
especially the crystallization behavior of the target proteins. In
addition, the effects of large fusion proteins are very much
contextual, and in some cases the net effect remains
controversial.5 This is because solubility cannot be controlled
without simultaneously changing other parameters, such as
precipitant type or concentration.11 Another recent strategy for
improving protein solubility, coined “supercharging”,36−38
consists of mutating surface hydrophobic residues to a charged
amino acid.4 However, supercharging can strongly affect the
activity and biochemical properties of the target proteins,
especially when they are driven by electrostatic interactions. On
the other hand, extremely short tags described in our present
and past studies8,9,19 can control protein solubility in a highly
versatile manner as they act as unstructured peptides and are
independent from the target protein, both from structural,
functional, and thermodynamic viewpoints. To date, the tag’s
solubilization effects were also demonstrated for a Gaussia
Luciferase, containing 10 disulfide bonded cysteines, which was
DOI: 10.1021/acs.cgd.5b00010
Cryst. Growth Des. 2015, 15, 2703−2711
Crystal Growth & Design Article

Figure 5. Effects of the SCP tags on protein structures and crystal packing. (a) Superimposed terminal regions (residues 55−64) of untagged BPTI-
19A (dark gray stick) and tagged variants (light gray stick). Panels b−h: Crystal asymmetric unit cell content and effects of peptide tags on crystal
packing. The asymmetric unit contents of untagged BPTI-19A (b; two monomers per unit cell, space group: C2) and its C5P (c; two monomers per
unit cell, space group: C2), C5S (d; two monomers per unit cell, space group: C2), C5N (e; three monomers per unit cell, space group: P6522),
C3R (f; four monomers per unit cell, space group: P21), C3K (g; eight monomers per unit cell, space group: P21) and C5H (h; eight monomers per
unit cell, space group: P21) tagged variants are shown. Figures were generated using Pymol graphics (www.pymol.org). In all panels a single chain is
shown in dark gray color.

2709 DOI: 10.1021/acs.cgd.5b00010

Cryst. Growth Des. 2015, 15, 2703−2711
Crystal Growth & Design
expressed in E. coli’s soluble fraction by attaching nine aspartic
acids to its C terminal.39
■ CONCLUSIONS
Protein solubility is a major concern in almost all aspects of
protein chemistry. Here, we demonstrated that short peptide
tags can be used for fine-tuning protein solubility without
affecting its other properties. This ability to control solely
protein solubility is especially important for analyzing and
possibly controlling protein crystallization. All tags that
produced variants with TS between 0.8 and 6 mg/mL
crystallized well. On the other hand, an Ile tagged BPTI
variant having extremely low solubility did not crystallize, as
expected, but also a highly soluble C5K variant did not
crystallize, and the crystals of other variants with transient
solubility >6 mg/mL did not diffract well either (too small or
low diffraction). Although the quantitative relationship between
crystallization behavior and the solubility parameters remains to
be fully determined, the present observations clearly suggest
some strong links between crystal quality and solubility.
■
*
ASSOCIATED CONTENT
S Supporting Information
Figure S1. Thermal stability of untagged and tagged BPTI-19A
variants in the absence and presence of ammonium sulfate
measured by circular dichroism. Figure S2. The effects of SCP
tags on protein solubility and crystallization behavior of model
BPTI-19A. Figure S3: Structures of the poly-amino-acid tags.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.cgd.5b00010.
Accession Codes
The coordinates and structure factors of BPTI-19A and its 5-
Ser, 5-Asn, 5-His, 5-Pro, 3-Arg and 3-Lys tagged variants have
been deposited in the Protein Data Bank under PDB IDs
3AUB, 3AUC, 3AUD, 3AUE, 3AUG, 3AUH, and 3WNY,
respectively.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: ykuroda@cc.tuat.ac.jp.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank Dr. Atsushi Kato for constructing and kindly
providing some of the tagged BPTI variant expression systems,
Patricia McGahan for English grammar editing, and members
of the Kuroda Laboratory for discussion. X-ray diffraction data
were recorded at the Photon Factory (PF, Tsukuba, Japan).
This work was supported by a Japan Society for the Promotion
of Science (JSPS) grant-in-aid for scientific research (KAKEN-
HI-21300110 and 15H04359) and a MEXT special priority
research area grant (KAKENHI-21107505) to Y.K. M.M.I.
acknowledges a JSPS postdoctoral fellowship, a JSPS Invitation
Fellowship Program for Research in Japan, and a Ministry of
Science and Technology (MOST), Bangladeshi fund.
■
ABBREVIATIONS
BPTI, bovine pancreatic trypsin inhibitor; BPTI-19A, a
simplified single-disulfide-bonded BPTI variant containing 19
alanines; DSC, differential scanning calorimetry; CD, circular
dichroism; RMSD, root mean square deviation; SCP tag,
2710
Article
Solubility Controlling Peptide tag; TS, transient solubility,
protein concentration in supernatant after 20 min of
incubation; LS, long-term solubility, protein concentration in
the supernatant after 48 h of incubation. Note: the expressions,
transient and long-term solubility are used according to ref 19,
where transient solubility does not imply that the system has
reached equilibrium. On the other hand, long-term solubility is
the concentration where equilibrium between amorphous
aggregation and the solution has been reached. Note also
that in ref 19 the solubilities are defined versus amorphous
aggregation rather than crystals.
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2711 DOI: 10.1021/acs.cgd.5b00010

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