Arg143 and Lys192 of the human mast cell chymase
mediate the preference for acidic amino acids in
position P2¢ of substrates
Mattias K. Andersson, Michael Thorpe and Lars Hellman
Department of Cell and Molecular Biology, Uppsala University, The Biomedical Center, Sweden
Introduction
Mast cells (MCs) are resident tissue cells that are dis-
tributed along the surfaces of the body. They are fre-
quently found in the mucosa of the airways and
intestine, in connective tissue of the skin, and around
blood vessels and nerves. Upon activation, MCs are
able to rapidly exocytose their cytoplasmic granules,
resulting in the release of prestored physiologically
active inflammatory mediators. The majority of pro-
teins found in these granules are serine proteases, and
one subfamily of these proteases comprises the
chymases. Chymases cleave substrates after aromatic
amino acids, and are therefore chymotrypsin-like.
Phylogenetic analyses of the chymases have led to
the identification of two distinct subfamilies, the a-chy-
mases and the b-chymases [1]. The a-chymases are
encoded by a single gene in all species investigated,
except for ruminants, where two very similar a-chym-
ase genes have been identified [2]. The b-chymases
Keywords
chymase; cleavage specificity; human
chymase; mast cell; site-directed
mutagenesis
Correspondence
L. Hellman, Department of Cell and
Molecular Biology, Uppsala University, The

targets for these enzymes.
Abbreviations
Ang, angiotensin; DC, dog chymase; EK, enterokinase; HC, human chymase; IPTG, isopropyl thio-b-
D-galactoside; MC, mast cell; mMCP,
mouse mast cell protease; OC, opossum chymase; rMCP, rat mast cell protease.
FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS 2255
have only been identified in rodents. Interestingly, the
rodent a-chymases mouse MC protease (mMCP)-5
and rat MC protease (rMCP)-5 have changed their pri-
mary cleavage specificity from aromatic amino acids
(chymotrysin-like) to aliphatic amino acids (elastase-
like).
A large number of in vitro substrates have been
identified for the chymases. However, the absolute
majority of these have never been shown to also be
substrates in vivo [3]. Therefore, the true functions of
the chymases most likely remain to be identified. In
order to increase our understanding of these enzymes,
a necessary step is to determine the most important
feature of an enzyme, the specificity-determining inter-
actions with its substrates.
In a previous study, we determined the cleavage
specificity in seven positions from positions P4 to P3¢
for human chymase (HC) [4]. The cleavage of the pep-
tide bond occurs between positions P1 and P1¢, where
the amino acids N-terminal of this bond are designated
P1, P2, P3, P4 Pn and those C-terminal P1¢,P2¢,
P3¢ Pn¢ [5]. The strongest preference observed,
besides the primary specificity for P1 Phe or Tyr, was
the preference for negatively charged (acidic) amino

b-chymases mMCP-1, rMCP-1 and rMCP-4 were found
to prefer other amino acids than Asp or Glu in this posi-
tion [6,12,13] (submitted manuscript Gallwitz et al.,
2009). When the amino acids in positions 40, 143 and
192 of the above chymases are compared, the five chy-
mases with a specificity for acidic P2¢ amino acids all
have Arg143 and Lys192. However, they differ at posi-
tion 40 (Table 1). Furthermore, none of the four
chymases that lack the acidic P2¢ specificity has an Arg
at position 143. However, three of them have Lys at
position 192. On the basis of these observations, we
hypothesized that Arg143 alone or in cooperation with
Lys192 mediates the preference for acidic amino acids
at position P2¢. In the present study, we tested the
roles of Arg143 and Lys192 as P2¢ specificity-determin-
ing residues. By in vitro mutagenesis, the HC coding
region was modified so that Arg143 was replaced by
Gln and Lys192 was replaced by Met, which are
amino acids found in the same positions of chymases
that lack acidic P2¢ specificity. Our results clearly show
that positions 143 and 192 have an effect in mediating
the acidic P2¢
specificity. Arg143 and Lys192 are essen-
tial in conferring a strong preference for acidic amino
acids at position P2¢ of the substrates.
Table 1. P2¢ specificity and amino acids found in positions 40, 143 and 192 of nine different chymases.
Chymase P2¢ specificity Residue 40 Residue 143 Residue 192 Reference
Human a-chymase Asp ⁄ Glu Lys Arg Lys 4
Opossum a-chymase Asp His Arg Lys 10
rMCP-5 (a-chymase) Glu > Leu Ser Arg Lys 11

purification, the three different recombinant HC
mutants were activated by removal of the His6-tag by
proteolytic cleavage with enterokinase (EK). Approxi-
mately 30 lg of each mutant was treated with EK for
5 h at 37 °C. Samples of inactive and activated prote-
ases were separated on SDS ⁄ PAGE gels, in order to
ensure successful removal of the His6-tag and the
EK-susceptible cleavage site (Fig. 1). Like the wild-
type enzyme, the mutated inactive proteases migrated
as 35 kDa bands, and the EK-digested enzymes
migrated as 33 kDa bands (Fig. 1). This is somewhat
over the theoretical value of 25 kDa for wild-type HC,
which indicates glycosylation at two sites of these
proteases. To purify the activated proteases from
contaminating serum and cellular proteins, imidazole,
and EK, they were purified over a heparin–Sepharose
column. The heparin–Sepharose-purified fractions were
separated on SDS ⁄ PAGE gels, and no contaminating
bands could be detected (Fig. 1). The proteolytic activ-
ities of the eluted fractions of the three mutated HCs
were analyzed by cleavage of the chymotrypsin-sensi-
tive chromogenic substrate S-2586 (MeO-Suc-Arg-
Ala-Tyr-pNA, Chromogenix, Mo
¨
lndal, Sweden, data
not shown).
Determination of the extended cleavage
specificity of the three HC mutants by phage
display technology
The phage library used to determine the extended

After the last biopanning, 44 individual phage
clones were isolated for each of the three HC
mutants, and the sequences encoding the randomly
synthesized nonapeptides were determined. The nucle-
otide sequences were then translated into nonapep-
tides, which were aligned on the basis of similarities
to the cleavage specificity of wild-type HC [4]. For
the Arg143 fi Gln mutant, 41 sequences were deter-
mined in total, two of which were obvious back-
ground sequences and therefore not included in the
–EK +EK Hep –EK +EK Hep –EK +EK Hep
R143Q K192M K192M
R143Q +
97
66
45
30
20
14
kDa
Fig. 1. Purification and activation of recombinant HC mutants. Three
different recombinant HC mutants were expressed with an N-termi-
nal His6-tag followed by an EK-susceptible sequence replacing the
signal peptide. These proenzymes were first purified on Ni
2+
–nitrilo-
triacetic acid beads ()EK), and then activated by removal of the
His6-tag by EK digestion (+EK). Following activation, the enzymes
were further purified on heparin–Sepharose columns (Hep). Proen-
zymes and activated enzymes before and after heparin–Sepharose

a clear overrepresentation of aliphatic amino acids,
particularly Val and Leu, was observed. In
position P1, Phe and Tyr were more frequently seen
than Trp. The three mutants also shared preferences in
the positions on the C-terminal side of the scissile
bond. The aliphatic Gly and Ala, and the hydrophilic
Ser, dominate in position P1¢. Similar preferences were
identified in position P3¢, where aliphatic amino acids
were generally preferred. However, in this position, the
hydrophilic amino acids Ser and Thr were also fre-
quently found. All of the positions mentioned above fit
very well with the cleavage specificity of wild-type HC,
as previously determined by our group [4]. The only
position where we detected an altered specificity
between the wild-type HC and the HC mutants was in
position P2¢. The strong preference for acidic amino
acids found in wild-type HC had disappeared in the
Arg143 fi Gln and Lys192 fi Met mutants and the
double mutant, and instead we observed a preference
for aliphatic amino acids. A comparison between the
wild-type HC and the HC mutants regarding the speci-
ficity for acidic amino acids at position P2¢ is shown in
Fig. 5. In a previous study, where the same phage-dis-
played nonapeptide library was used to determine the
cleavage specificity of wild-type HC, a negatively
charged amino acid at position P2¢ was seen in 58% of
the sequences. For the Arg143 fi Gln mutant, this
figure was reduced to 25%, and for the Lys192 fi Met
mutant, 19% of the sequences were aligned with an
acidic amino acid at position P2¢. When these muta-

20
40
60
80
100
0123456
Fig. 2. Amount of released T7 phages after digestion with HC
mutants, as compared with an NaCl ⁄ P
i
control. A library of ran-
domly synthesized nonamers expressed at the C-terminus of T7
phages were subjected to cleavage by HC mutants. Selection for
nonamers susceptible to cleavage by the HC mutants was per-
formed in five or six rounds of selection (biopannings). After each
biopanning, the amount of released phages was determined and
compared to an NaCl ⁄ P
i
control. The ratio of phages released by
enzyme digestion over the NaCl ⁄ P
i
control for each biopanning is
shown. The Arg143 fi Gln mutant is indicated by a dashed line,
the Lys192 fi Met mutant by a dotted line, and the
Arg143 fi Gln + Lys192 fi Met double mutant by an unbroken
line.
P2¢ specificity of the human mast cell chymase M. K. Andersson et al.
2258 FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS
was added to the C-terminus of this protein (Fig. 6A).
A number of related and unrelated substrate sequences
were also produced with this system, by ligating the

R R
V
G
V
M
F
S S
V
A A
V
GP
F
S SM M
A
L L
E
Y
V
G
I
K
G
S
IL
F
G G
S
V
A
R

FF
T
L
M
G
S
L
R
F
LV
T
W
G
T
P A
FF
LV
A G
D
G
V
F
T
LV
E
W
A A
I
FF
V

R
L
R R
F W
V
S
R
V
A
I
P
F
A
LV
F
G
E
S
A
F F
G
S
V
G
T
L
R
H
W WW
RR

T
A
F Y Y F
G
LI
GA P
2
Y WF
L
T
G A
T
G
W
H H H
L L
S S
G
E
KR
P4 P3 P2 P1 P1′P2′ P3′
R143Q + K192M
R
I
S
F
A G
E
V
F

T
D
M
KR
V
G
W
PP G G
Y
I
G
R
L
W
R
L
Y
S
L
E
L
T
V
F
K
W
E
K
I
P

W
FF
S
L
T
L
A
L
M
V
T
L
F W
G
T
P A
Y
S
A
S
V V
W
G
E
S
F
S H H
L V
P
Y

E
A
Y
S
Y
M
L
R
F
A
L
P
F
A
W
G
L
Y
G
D
L
F
V
T
A
W
F
E
A
L

S
F
A
WW
G
H
Y
E
A
F
LL
W
A
V
FF
S
W
V
A
V
S
P
F
A
L
H
L
W
P
YW

S
L
M
P
P4 P3 P2 P1 P1′P2′ P3′
K192M
Negatively charged
Positively charged
Aromatic
Large aliphatic
Small aliphatic
P G G
W
S
R
M
L LV
R
P
VV L
F
S T
G
E
S
E
MT
I
T
V

S
A
D
I
G
M
P
Y
S T
L
M
F
G
W
A
S
VV
F
G
T T
Y F
MM S
G
VL V
V
Y
V
M
G
S

P A
D
I
W
E
W
P
L
F
L
A A
W
VV
S
F W
V
A
D
T
W W
R
D
F
S T N
G
W
A A
Y F
L
D

Y
D
4
2
Y
GG
E
S
VV LV
P4 P3 P2 P1 P1′P2′ P3′
R143Q
ABC
Fig. 3. Phage-displayed nonamers susceptible to cleavage by HC mutants after five or six biopannings. After the last selection step, phages
released by proteolytic cleavage of the HC mutants were isolated, and the sequences encoding the nonamers were determined. The general
sequence of the T7 phage capsid proteins is PGG(X)
9
HHHHHH, where (X)
9
indicates the randomized nonamers. The protein sequences were
aligned into a P4–P3¢ consensus, where cleavage occurs between positions P1 and P1¢. If the sequence was found more than once, this is
indicated by the corresponding number to the left of the sequence. The amino acids are color coded according to the side chain properties
as indicated in the key. For the Arg143 fi Gln mutant (A), 24 unique sequences were aligned; for the Lys192 fi Met mutant (B), 36 unique
sequences were aligned; and for the Arg143 fi Gln + Lys192 fi Met double mutant (C), 32 unique sequences were aligned. The
sequences with one aromatic amino acid (potential cleavage site) are placed on the top, followed by sequences containing two, three or four
aromatic amino acids.
M. K. Andersson et al. P2¢ specificity of the human mast cell chymase
FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS 2259
0
10
20

30
40
FYWGAVL I PSTCMNQHKRDE
0
10
20
30
40
FYWGAVL I PSTCMNQHKRDE
0
10
20
30
40
FYWGAVL I PSTCMNQHKRDE
0
10
20
30
40
FYWGAVL I PSTCMNQHKRDE
WT
R143Q
K192M
R143Q+
K192M
P3
0
10
20

30
40
FYWGAVL I PSTCMNQHKRDE
0
10
20
30
40
FYWGAVL I PSTCMNQHKRDE
0
10
20
30
40
FYWGAVL I PSTCMNQHKRDE
0
10
20
30
40
FYWGAVL I PSTCMNQHKRDE
WT
R143Q
K192M
R143Q+
K192M
P3´
0
10
20

30
40
FYWGAVL I PSTCMNQHKRDE
0
10
20
30
40
FYWGAVL I PSTCMNQHKRDE
0
10
20
30
40
FYWGAVL I PSTCMNQHKRDE
0
10
20
30
40
FYWGAVL I PSTCMNQHKRDE
WT
R143Q
K192M
R143Q+
K192M
P1´
0
10
20

WT
0
10
20
30
40
50
60
70
FYWGAVL I PSTCMNQHKRDE
K192M
P1
Occurrence (%)
Occurrence (%)Occurrence (%)
Occurrence (%)Occurrence (%)Occurrence (%)Occurrence (%)Occurrence (%)Occurrence (%)Occurrence (%)
Fig. 4. Distribution of amino acids at positions P4–P3¢ in phage-displayed nonamers cleaved by wild-type (WT) HC or HC mutants after five
or six biopannings. On the basis of the alignment in Fig. 3 and previously published data on wild-type HC, the percentage of each amino acid
present in each position, P4 to P3¢, was calculated. The amino acids are ordered from left to right: aromatic, aliphatic, hydrophilic, basic (pos-
itively charged), and acidic (negatively charged).
P2¢ specificity of the human mast cell chymase M. K. Andersson et al.
2260 FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS
efficiently than the HC consensus sequence, whereas
the HC double mutant cleaved this substrate almost as
efficiently as its own consensus site (Fig. 6B,C).
A few additional substrates were also included in
this study. The optimal sequence for cleavage by OC
has recently been determined [10]. As compared with
HC, this enzyme was found to have a preference for
Trp over Phe and Tyr at position P1. When we ana-
lyzed the cleavage of this sequence (VGLWLDRV), we

seen when Arg226 was replaced by Glu in the same
enzyme [16]. In the present study, we used the same
strategy to investigate the effect of two amino acids on
the extended substrate interactions of HC. We showed
that we could change the cleavage specificity of HC
for position P2¢ of substrates by mutating posi-
tions 143 and 192 in the enzyme. By replacing Arg143
by Gln or Lys192 by Met, which are amino acids com-
monly found in these positions in related rodent chy-
mases, the preference for acidic amino acids at
position P2¢ was markedly reduced, whereas the cleav-
age specificity for all other positions was essentially
unaffected. The basic amino acids at positions 143 and
192 attract and stabilize the interaction of acidic amino
acid side chains at position P2¢. When either of these
positively charged residues was replaced by an amino
acid with an uncharged side chain, a preference for
mainly aliphatic amino acids was observed. However,
a weak preference for acidic P2¢ amino acids still
remained for the two single mutants. In the double
mutant, the preference for negatively charged P2¢
amino acids was lost completely.
In order to put our hypothesis to a stringent test, we
aligned the enzyme-selected peptides with acidic P2¢
amino acids, when an aromatic amino acid could be
aligned at position P1. We then aligned the sequences
according to the cleavage specificity of the wild-type
enzyme, considering the remaining positions. We could
thus be certain that we were not overestimating the
effect of the mutations. However, a large fraction of

the Arg143 fi Gln mutant, the Lys192 fi Met mutant, and the
Arg143 fi Gln + Lys192 fi Met double mutant. Glu residues in
this position are depicted as open bars, and Asp residues as filled
bars. The occurrence of acidic amino acids in position P2¢ of wild-
type HC was determined in a previous study [4].
M. K. Andersson et al. P2¢ specificity of the human mast cell chymase
FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS 2261
A
B
C
D
E
F
P2¢ specificity of the human mast cell chymase M. K. Andersson et al.
2262 FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS
conclude from our data that the preference of HC for
acidic amino acids at position P2¢ is mediated by the
combined effects of both Arg143 and Lys192. A simi-
lar combined effect has, to our knowledge, never been
identified for a serine protease.
By the use of a new type of recombinant substrate,
we were also able to verify this marked change in
preference for a negatively charged amino acid at
position P2¢ by mutating Arg143 and Lys192. Wild-
type HC was found to cleave the consensus substrate
four-fold to five-fold more efficiently than the substrate
in which the amino acid at position P2¢ had been
exchanged for a Gly. In contrast, the HC double
mutant showed a two-fold to five-fold greater prefer-
ence for the substrate in which position P2¢ was not

ence for acidic P2¢ amino acids. The gerbil chymase-2
also has Lys143 and Lys192, and may therefore
also have a lower preference for acidic amino acids at
position P2¢.
HC efficiently converts Ang I to Ang II (cleavage of
the Phe8-His9 bond of Ang I), and the structural
requirements for this specificity have been addressed in
several studies. Synergistic interactions of posi-
tions P4–P1, together with the dipeptidyl leaving group
of Ang I, were found to be important for efficient con-
version by HC [18,19]. However, the side chains on the
leaving group of Ang I do not seem to be important
for the selectivity of HC in converting Ang I [19].
Instead, the negatively charged C-terminal carboxyl
group of Ang I probably interacts with the Lys40 side
chain of HC to stabilize the substrate [8]. Lys40 and
Arg143 of HC have previously been analyzed for their
role in the selective Ang I conversion by HC. Analysis
of Lys40 fi Ala and Arg143 fi Gln mutants of HC
showed that Lys40 but not Arg143 contributed to the
high specificity of HC in converting Ang I to Ang II
[20]. The Arg143 fi Gln mutant was actually shown to
be more active than the wild type in converting Ang I,
indicating a minor role or no role at all of Arg143 in
Ang I conversion. The lack of function for Arg143
and Lys192 in Ang I conversion is also substantiated
by the fact that mMCP-1, which has Lys143 and
Met192, and has a P2¢ specificity for Leu, is a good
Ang I converter [21]. Furthermore, the rat vascular
chymase, which has Arg143 and Thr192 and thus does

other than Ang I are evolutionarily conserved targets
for the MC a-chymases or their rodent counterpart,
the b-chymase mMCP-4.
The search for potential in vivo substrates is being
performed using bioinformatic screening. However, the
identification of these substrates may be challenging,
mainly because of the ability of HC to interact with
different amino acid side chains in each subsite of the
enzyme, which leads to a very large number of poten-
tial substrates during the screening of the full human
proteome. This highlights the importance of factors
other than the extended cleavage specificity in deter-
mining whether a protein will be a biologically signifi-
cant substrate for HC. For example, the local
concentration of the protease and availability of the
potential substrate in the immediate environment are
significant factors. The extended cleavage specificity
predicts those sequences (and hence substrates) that
would be preferentially cleaved within a shorter time
frame. These substrates can theoretically be cleaved
before the protease encounters a protease inhibitor.
The influence of the cleavage sequence position in the
protein is also important. It is likely that surface-
exposed, flexible regions would be cleaved efficiently.
Conversely, nonexposed, more rigid regions would
remain uncleaved, regardless of whether the preferred
sequence for the protease was present or not. In the
event of substrate cleavage, whether this leads to a bio-
logical effect also needs to be determined. With knowl-
edge of the extended cleavage specificity and other

antisense primer, 5¢-CCAGAGTCTCCC
ATAAATGCAGA
TTTTGTCTTCCTG-3¢. These primers had a melting tem-
perature of 78 °C, and the mismatches resulting in replace-
ment of the Lys codon with a Met codon are underlined.
The Arg143 fi Gln + Lys192 fi Met double mutant was
produced by introducing the Lys192 fi Met mutation into
the Arg143 fi Gln mutant. All primers were purchased
from Sigma-Aldrich (Steinheim an Albuch, Germany) in a
PAGE-purified form. Thermal cycling was performed with
PfuUltra high-fidelity DNA polymerase (provided by the
manufacturer). After this PCR step, nonmutated parental
DNA constructs were digested with Dpn1 endonuclease for
1 h at 37 °C. The remaining nondigested and mutated DNA
vector constructs were ethanol precipitated. The salt and
ethanol concentration during precipitation was 75 mm
NaAc (pH 5.2) in 75% ethanol. After precipitation, the
DNA was resuspended in 15 lL of double-distilled H
2
O.
This DNA was then used to transform XL10-Gold ultra-
competent E. coli cells (provided by the manufacturer). All
mutants were sequenced to confirm the inserted mutations
and the absence of unintended additional mutations, by
using an ABI PRISM 3730 DNA Analyzer (Applied Biosys-
tems, Foster City, CA, USA) and vector-specific primers.
Production and purification of recombinant HC
mutants
The vector constructs encoding the HC mutants were trans-
fected into a human embryonic kidney cell line (HEK 293

tic acid beads were washed five times with washing buffer
(1 m NaCl, 0.2% Tween in NaCl ⁄ P
i
). Bound protein was
then eluted with elution buffer (100 mm imidazole, 0.2%
Triton X-100 in NaCl ⁄ P
i
). Protein purity and concentration
was estimated by separation on 12.5% SDS ⁄ PAGE gels.
Protein samples were mixed with sample buffer, and b-mer-
captoethanol was added to a final concentration of 5%. To
visualize the protein bands, the gel was stained with Coo-
massie Brilliant Blue.
Activation and further purification of
recombinant HC variants
Approximately 30 lg of each HC mutant was diluted 1 : 2
in double-distilled H
2
O and digested for 5 h at 37 °C with
EKMax EK (Invitrogen), using one unit per 10 lgof
recombinant protease.
In order to remove EK and other impurities, the
EK-digested HC mutants were purified by affinity chroma-
tography on heparin–Sepharose columns as described pre-
viously [13]. PolyPrep Chromatography columns
containing 0.2 mL of heparin–Sepharose beads (Sigma-
Aldrich) were equilibrated with NaCl ⁄ P
i
(pH 7.2). Each
EK-cleaved HC mutant was applied to a column, and this

Ni
2+
–nitrilotriacetic acid beads by their His6-tags for 1 h
at 4 °C under gentle agitation. Unbound phages were
removed by washing 10 times in 1.5 mL of 1 m NaCl and
0.1% Tween-20 in NaCl ⁄ P
i
(pH 7.2), and two subsequent
washes with 1.5 mL of NaCl ⁄ P
i
. The beads were finally
resuspended in 1 mL of NaCl ⁄ P
i
. Activated and heparin–
Sepharose-purified HC mutant ( 0.1 l g) was added to the
resuspended beads and left to digest susceptible phage no-
napeptides under gentle agitation at room temperature
overnight. NaCl ⁄ P
i
without protease was used as control.
Phages with a random peptide that was susceptible to pro-
tease cleavage were released from the Ni
2+
–nitrilotriacetic
acid matrix, and the supernatant containing these phages
was recovered. To ensure that all of the released phages
were recovered, the beads were resuspended in 100 lLof
NaCl ⁄ P
i
(pH 7.2) and the supernatant, after mixing and

brary for 1 h at 4 °C under gentle agitation, the Ni
2+
–
nitrilotriacetic acid beads were washed 15 times in 1.5 mL
of 1 m NaCl and 0.1% Tween-20 in NaCl ⁄ P
i
(pH 7.2), and
then twice in 1.5 mL of NaCl ⁄ P
i
.
Following five or six rounds of selection, 44 plaques for
each HC mutant were isolated from LB plates after plating
in top agarose. Each phage plaque, corresponding to a
phage clone, was dissolved in phage extraction buffer
(100 mm NaCl and 6 mm MgSO
4
in 20 mm Tris ⁄ HCl,
pH 8.0) and vigorously shaken for 30 min in order to
extract the phages from the agarose. The phage DNA was
then amplified by PCR, using primers flanking the variable
region of the gene encoding the modified T7 phage capsid
protein. After amplification, PCR fragments were purified
using the E.Z.N.A Micro Elute Cycle-Pure kit (Omega bio-
tek, Doraville, GA, USA). Purified PCR fragments were
then sequenced (Macrogen Inc., Seoul, Korea) using an
ABI PRISM 3730 DNA Analyzer (Applied Biosystems).
M. K. Andersson et al. P2¢ specificity of the human mast cell chymase
FEBS Journal 277 (2010) 2255–2267 ª 2010 The Authors Journal compilation ª 2010 FEBS 2265
Generation of a consensus sequence from
sequenced phage inserts

cloning by sequencing of both DNA strains. The plasmids
were then transformed into the E. coli Rosetta gami strain
for protein expression (Novagen; Merck, Darmstadt,
Germany). A 10 mL overnight culture of the bacteria har-
boring the plasmid was diluted 10 times in LB + ampicil-
lin, and grown at 37 °C for 1–2 h until D
600 nm
reached 0.5.
IPTG was then added to a final concentration of 1 mm.
The culture was then grown at 37 °C for an additional 3 h
with vigorous shaking, after which the bacteria were pel-
leted by centrifugation at 1600 g for 12 min. The pellet was
washed once with 25 mL of NaCl ⁄ P
i
+ 0.05% Tween-20.
The pellet was then dissolved in 2 mL of NaCl ⁄ P
i
and soni-
cated six times for 30 s each to open the cells. The lysate
was centrifuged at 10 000 g for 10 min, and the supernatant
was transferred to a new tube. Five hundred microliters of
Ni
2+
–nitrilotriacetic acid slurry (50 : 50) (Qiagen, Hilden,
Germany) was added, and the sample was slowly rotated
for 45 min at room temperature. The sample was then
transferred to a 2 mL column, and the supernatant was
allowed to slowly pass through the filter, leaving the Ni
2+
–

ume of 2 · sample buffer. One microliter of b-mercaptoeth-
anol was then added to each sample, and this was followed
by heating for 3 min at 80 °C. Twenty microliters from
each of these samples was then analyzed on 4–12% precast
SDS ⁄ PAGE gels (Invitrogen). The gels were stained over-
night in colloidal Coomassie staining solution, and
destained for several hours according to previously
described procedures [25]. The intensities of the individual
bands on the gel were determined from scanned high-reso-
lution pictures by densitometric scanning of the gels and
using imagej (rsb.info.nih.gov ⁄ nih-image ⁄ ). In order to
obtain good estimates of the differences in activity towards
different substrates, different concentrations of the enzyme
were used in several individual experiments. The combined
results from these different gels were then used to obtain a
good estimate of the differences in activity against the vari-
ous substrates.
Acknowledgement
This study was supported by grants from the Swedish
National Research Council (VR-NT).
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