C-Terminal extension of a plant cysteine protease
modulates proteolytic activity through a partial inhibitory
mechanism
Sruti Dutta, Debi Choudhury, Jiban K. Dattagupta and Sampa Biswas
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India
Keywords
C-terminal extension; cysteine proteases;
modulation of proteolytic activity;
papain-like; thermostable
Correspondence
S. Biswas, Crystallography and Molecular
Biology Division, Saha Institute of Nuclear
Physics, 1 ⁄ AF Bidhannagar, Kolkata
700 064, India
Fax: +91 332 337 4637
Tel: +91 332 337 5345
E-mail: [email protected]
(Received 14 March 2011, revised 16 May
2011, accepted 22 June 2011)
doi:10.1111/j.1742-4658.2011.08221.x
The amino acid sequence of ervatamin-C, a thermostable cysteine protease
from a tropical plant, revealed an additional 24-amino-acid extension at its
C-terminus (CT). The role of this extension peptide in zymogen activation,
catalytic activity, folding and stability of the protease is reported. For this
study, we expressed two recombinant forms of the protease in Escherichia
coli, one retaining the CT-extension and the other with it truncated. The
enzyme with the extension shows autocatalytic zymogen activation at a
higher pH of 8.0, whereas deletion of the extension results in a more active
form of the enzyme. This CT-extension was not found to be cleaved during
autocatalysis or by limited proteolysis by different external proteases.
Molecular modeling and simulation studies revealed that the CT-extension

+CT
,
recombinant mature ervatamin-C with C-terminal extension; rmErv-C
DCT
, recombinant mature ervatamin-C without C-terminal extension;
rproErv-C
+CT
, recombinant proervatamin-C with C-terminal extension; rproErv-C
DCT
, recombinant proervatamin-C without C-terminal
extension.
3012 FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS
pro-region and a mature catalytic domain [2–4]. Fol-
lowing synthesis, the pre-peptide is removed during
passage to the lumen of the endoplasmic reticulum [2].
The inactive proenzyme subsequently undergoes prote-
olytic processing to produce an active mature enzyme
by autocatalytic cleavage of the propeptide part at the
N-terminus [2]. It is known that the propeptide at the
N-terminus of the protease acts as an intramolecular
chaperone to mediate correct folding of the protease
[2]. The mature catalytic domain of the enzyme of this
family has a molecular mass of  21–30 kDa and
shares a common fold with papain, the archetype
enzyme of the family. These proteases are folded into
two compact interacting domains of comparable size,
delimiting a cleft which contains the active site residues
cysteine and histidine, forming a zwitterionic catalytic
dyad (Cys
)

mania pifanoi, plays a role in the regulation of enzyme
activity [11]. CT-extension in mammalian and yeast
bleomycin hydrolase [12,13] is a key factor which regu-
lates their endo-peptidase or exo-aminopepetidase
activity by blocking the unprimed subsites in the
enzymes.
Ervatamin-C (Erv-C) is a papain-like cysteine prote-
ase (
EC 3.4.22) with high stability purified from the
latex of a tropical plant Ervatamia coronaria [14]. The
3D structure of Erv-C reveals an extra disulfide bond,
shorter loop regions and additional electrostatic inter-
actions in the interdomain space, which are thought to
be responsible for its high stability [15]. Sequencing
of the cDNA (from mRNA) of Erv-C from the leaf
of the plant in our laboratory [16], and comparison of
the cDNA-derived amino acid sequence with other
members of the family reveal that Erv-C is synthesized
as a precursor protein and in addition to the pre- (19
amino acids), pro- (114 amino acids) and mature (208
amino acids) parts, it contains an extension of 24
amino acids at the CT of the mature enzyme [16]
(Fig. 1A). This CT-extension was not observed, how-
ever, when the mature Erv-C was purified directly
from the latex of the plant [15].
In this article, we attempt to understand the role of
this CT-extension in zymogen activation, enzyme activ-
ity, folding and stability in vitro at the molecular level
from structural and functional points of view.
Results

buffer, pH 8.00, at 60 °C for 25–30 min, whereas the
purified refolded rproErv-C
DCT
could be converted
into its mature form (rmErv-C
DCT
) by the same activa-
tor in 50 mM Na-acetate buffer, pH 4.5, at 60 °C for
45 min (Fig. 2A). Thus the zymogen activation process
occurs at different pH and time of maturation for the
two enzymes. The molecular mass of the mature
enzyme rmErv-C
+CT
is higher ( 27 kDa) than that
of rmErv-C
DCT
( 25 kDa) as observed in the
SDS ⁄ PAGE analyses (Fig. 2A). This difference in
S. Dutta et al. Role of C-terminal extension in a cysteine protease
FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS 3013
molecular mass almost fits the theoretically calculated
value for the 24-amino-acid CT-extension. We
expected, therefore, that the CT-extension continued to
remain attached with the mature enzyme even after
autocatalytic processing of rproErv-C
+CT
. Gelatin gel
assay (Fig. 2B) and western blot analyses (Fig. 2C)
also confirmed the retention of the CT-extension in the
mature rmErv-C

114 aa
208 aa
BamH1
34 aa
Start
Stop
Xho1
SS SS
G
SSS
S
N-Pro
356 aa
III
MSTLFII
S
ILLFLAS
F
SYAMDI
S
TIEYKYDKSS
AWRTDEEVKEIYELWLAKHDKVY
SG
LVEYEKRFEIFKDNLKFIDEH
NSENHTYKMGLTPYTDLTNEEFQAIYLGTRSDTIHRLKRTINISERYAYEAGDNLPEQIDWRKKGAVTPVKNQGKCG
SCWAFSTVSTVESINQIRTGNLISLSEQQLVDCNKKNHG
CKGGAFVYAYQYIIDNGGIDTEANYPYKAVQGPCRAAK
KVVRIDGYKGVPHCNENALKKAVASQPSVVAIDASSKQF
QHYKSGIFSGPCGTKLNHGVVIVGYWKDYWIVRNSW
GRYWGEQGYIRMKRVGGCGLCGIARLPYYPTKA

in vitro using seven different proteases
(four cysteine proteases Erv-A, -B, -C and papain from
the plant latex; two serine proteases trypsin and chy-
motrypsin from bovine pancreas; one aspartic protease
pepsin from porcine stomach mucosa), each having
sequences specific for their cleavage in the amino acid
sequence of the CT-extension. The activated mature
protein thus generated in each case shows a band at
the same position ( 27 kDa) like that in the autoacti-
vated enzyme, as observed in SDS ⁄ PAGE analyses
(Fig. S1), indicating that these external enzymes can
not cleave the CT-extension. Even with a prolonged
digestion time (24 h), the same result was obtained for
all proteases except trypsin (Fig. S1). Trypsin digestion
for 24 h resulted in a truncated protein at  26 kDa,
slightly above the activated mature rmErv-C
DCT
( 25 kDa). This result probably indicates that trypsin
has some accessibility to its sites of specificity (Lys,
which is in position 7 of the CT-extension) (Fig. S2)
and only after a prolonged incubation time can it
result in a band at a slightly lower molecular mass
position, as observed in the SDS ⁄ PAGE analysis
(Fig. S1).
Specific activity and optimum temperature of
activity
The optimum temperature of activity, T
opt
(Fig. 3), for
both forms is 65 °C. Interestingly, it was observed that

substrate with a valine at the P2 position which is
known to act as a substrate for Erv-C [17]. The kinetic
constants of the two recombinant enzymes (Table 1)
clearly show that rproErv-C
DCT
has almost 10 times
97 kDa
66 kDa
66 kDa
45 kDa
36 kDa
29 kDa
24 kDa
20 kDa
43 kDa
rproErv-C
+CT
rproErv-C
+CT
rproErv-C
ΔCT
rmErv-C
+CT
rmErv-C
ΔCT
rproErv-C
ΔCT
rmErv-C
ΔCT
rmErv-C

, rproErv-C
DCT
, rproErv-C
+CT
and rproErv-C
DCT
to rproErv-C
+CT
,
rproErv-C
DCT
, rmErv-C
+CT
and rmErv-C
DCT
’].
S. Dutta et al. Role of C-terminal extension in a cysteine protease
FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS 3015
higher activity than rproErv-C
+CT
. One can probably
conclude that the CT-extension has some inhibitory
effect on the activity of the enzyme against a small
peptide.
Thermal stability
Temperatures of maximum proteolytic activity (T
max
)
for rproErv-C
+CT

90
80
70
60
Residual activity (%)
Temperature (°C)
50
40
30
20
10
0
30 35 40 45 50 55 60 65 70 75 80 85 90 10095
Fig. 3. Determination of optimum temperature of activity (T
opt
)of
rproErv-C
+CT
and rproErv-C
DCT
. Purified proenzymes (10–20 lg)
were converted to their respective mature forms and the percent-
age residual enzyme activities were determined with respect to the
maximum activity using an azocasein assay at different tempera-
tures, as described in Materials and methods. Each data point is an
average of three independent experiments having similar values
(Table S3).
Table 1. Kinetic constants using the substrate N-benzoyl-Phe-Val-Arg-pNA. Specific activity using azocaesin and IC
50
value for the inhibitor

)1
)
IC
50
against
E-64 (n
M)
c
rproErv-C
+CT
0.0170 ± 0.004 88.33 ± 56.77 0.193 No activity 15.87 ± 1.36 482.5 ± 108.0
rproErv-C
DCT
0.2295 ± 0.057 127.3 ± 48.46 1.803 13.21 ± 2.32 35.27 ± 1.78 349.1 ± 62.0
Latex Erv-C [17] 9.312 1063 8.76 75.0 ND 225.0
a
Given standard errors were calculated based on nonlinear fitting of the Michaelis–Menten saturation curve using the software Graphpad
PRISM (http://www.graphpad.com/prism).
b
Each value of specific activity of rproErv-C+CT and rproErv-CDCT is a mean of three independent
experiments ± SD.
c
Given standard deviations were calculated from linear regression plot of residual activity and inhibitor concentration.
Table 2. Kinetic stabilities. ND, not determined.
T
max
(°C) T
50
(°C)
t

+CT
and rpr-
oErv-C
DCT
. Each purified proenzyme (10–20 lg) was treated for
10 min at different temperatures followed by activation of the pro-
proteins to their respective mature forms. The percentage residual
enzyme activities (at each temperature) were determined with
respect to the maximum activity using an azocasein assay at 65 °C
as described in Materials and methods. Each data point is an aver-
age of three independent experiments having similar values
(Table S3).
Role of C-terminal extension in a cysteine protease S. Dutta et al.
3016 FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS
performed. The total energy of the whole system and
root mean square deviation (RMSD) from the starting
structure are essential to determine the sustainability
and convergence of MD simulation. Figure 6A shows
the RMSD of backbone atoms of the CT-extension in
association with a mature domain as well as in an iso-
lated form. The graph shows that the RMSD reached
below  0.4 A
˚
when the extension is attached to the
mature domain and is > 1 A
˚
when the extension is on
its own. The fluctuation in the radius of gyration was
also analyzed (Fig. 6B) as a measurement of the over-
all stability of the CT-extension for both forms. These

+CT
) reveals that the Leu of
100
rproErv-C
+CT
rproErv-C
ΔCT
90
80
70
60
Residual activity (%)
Time in min
50
40
30
20
10
0
0
10
20
30
40
50
60
120
180
240
300

a minimum distance of 1.9 A
˚
between two atoms of
the two leucines (Fig. 8A). Because the Leu23 residue
of the CT-extension of the rmErv-C
+CT
molecule is
buried inside the S2 pocket, it restricts access of Leu at
the P2 position of E-64 and results in a higher IC
50
value (482.5 nM) of E-64 inhibition compared with
rmErv-C
DCT
(349.1 nM) (Table 1) in which the exten-
sion is truncated. Modeling studies also reveal that
although this CT-extension blocks some of the
unprimed subsites beyond S2, access to the catalytic
centre (Cys-His dyad) is not totally blocked.
Discussion
It is known that the autocatalytic processing of
papain-like cysteine proteases from pro- to mature
form generally occurs at acidic pH [18]. The 3D struc-
tures of papain-like cysteine proteases in the pro-form
[19–23] reveal that N-terminal propeptide part adopts
a specific globular structure which is conserved among
the family despite a relatively low homology in their
amino acid sequences. Previous reports suggest that an
acidic pH induces a conformational change in the
N-terminal propeptide domain, resulting in a molten
globule state and thereby the activation process is trig-

CT-extension contains six negatively charged residues
(one aspartate and five glutamates) (Fig. 1A) and our
previous molecular modeling studies [16] indicated that
this negatively charged region of the C-terminus could
A
B
Fig. 8. (A) The structure of the catalytic cleft and unprimed sub-
sites region of mature Erv-C with modeled CT-extension. The
mature catalytic domain is represented as an electrostatic potential
surface and the CT-extension as a stick model with C atoms in
green. The inhibitor E-64 (taken from the structure of Erv-C and
E-64 complex; Protein Data Bank ID
2PRE) is also docked for com-
parison and is represented as a stick model with C atoms in brown.
The catalytic cysteine residue (C25) of Erv-C is represented as a
ball and stick model (light blue) and the S2 subsite residues are rep-
resented as a stick model (light pink). The minimum distance
(1.9 A
˚
) between P2 (Leu23) of the CT-extension and P2(Leu) of
E-64 are indicated by the dotted line. (B) The last four residues,
F21P22L23A24, of the CT-extension domain (C atoms are shown in
magenta) and the neighboring residues of Erv-C mature domain
within 4.2 A
˚
(C atoms are shown in deep bottle green). Interdomain
distances within 4.2 A
˚
are shown in green.
Role of C-terminal extension in a cysteine protease S. Dutta et al.

in vivo [9,10]. So, in this study, we performed an
in vitro transactivation experiment using three latex
enzymes Erv-A, -B and -C from the same plant, and
papain from papaya latex and two other serine (tryp-
sin and chymotrypsin) and one aspartic (pepsin) prote-
ases. The results showed that this extension is also not
cleaved by any of these proteases in their optimum
condition of activity. However, because no experiment
has been carried out in vivo, the possibility of intermo-
lecular cleavage of the CT-extension by a different
vacuolar protease under specific in vivo conditions can
not be ruled out.
The results of enzyme kinetic studies show that the
proteolytic activities of the two recombinant forms
(rproErv-C
+CT
and rproErv-C
DCT
) are not of the same
order (Table 1). In optimum temperature determina-
tion using azocasein as a substrate, rmErv-C
+CT
does
not show any proteolytic activity when activity is
assayed below 45 °C, but activity increases suddenly
beyond 60 °C and sufficient activity is retained in the
temperature range 65–75 °C (Fig. 3) with the highest
being at 65 °C. This activity profile is different for
rmErv-C
DCT

protein substrate. But at a higher temperature (beyond
60 °C), this interference is partly removed and the
enzyme can show proteolytic activity to the protein
substrate. This behavior of the enzyme differs for a
smaller peptide substrate, where the enzyme can work
on this peptide even at lower temperatures, although
to a lesser extent. Molecular modeling studies show
that the C-terminal tail blocks the unprimed subsites
beyond S2 of the enzyme thus inhibiting the endopep-
tidase activity at temperatures below 55 °C for azocae-
sin. Perhaps, at a higher temperature, a reduction of
the association affinity for this tail towards the
unprimed subsites occurs leaving the subsites partially
free for substrate binding. This is in conformity with a
report [25] that the CT-extension sometimes has an
inhibitory property towards some enzymes. Blocking
of unprimed subsites is also found in other papain-like
proteases by different mechanisms either by a mini-
chain like cathepsin-H [26] or an exclusion domain in
cathepsin-C [27] or by C-terminal extension in bleomy-
cin hydrolase [12,13]. In all these cases, each protease
loses its endo-peptidase activity and functions as an
aminopeptidase because their unprimed subsites are
blocked as found here. A superposition of the struc-
tures of cathepsin-H and bleomycin hydrolase on the
modeled structure of rmErv-C
+CT
revealed that
although the blocking strategy of these peptides is sim-
ilar, in the first two proteases the blocking peptides

We have noted that both recombinant protease
forms can fold efficiently in vitro. So the CT-extension
does not appear to have any noticeable effect on proper
folding of the protein in vitro. However, our observa-
tions also indicate that this extension has some effects
in the maturation process and activity of the protease.
It suggests two possibilities for the presence of the CT-
extension in the enzyme: either it carries the signal for
vacuolar sorting and after that is cleaved by another
enzyme in vivo, or an mRNA (cDNA) exists in the leaf
of the plant that essentially encodes an isoform of Erv-
C which is present in the latex of the plant.
Materials and methods
Cloning, expression, purification and refolding of
rproErv-C
+CT
The entire open reading frame of the Erv-C precursor had
been cloned previously in our laboratory in pTZ57R ⁄ T
vector [16]. A fragment of the original cDNA encoding the
prodomain, the mature domain and CT-extension of Erv-C
was PCR-amplified from this clone using primers (Forward:
5¢-CCC
GGATCCATGGACATATCTACC-3¢ and Reverse:
5¢-GGT
CTCGAGTTAAGCAAGTGGAAAAGCT-3¢)desig-
ned to delete the pre-peptide (the signal peptide) and to
include the restriction sites for BamHI and Xho1 (under-
lined) to facilitate cloning into pET-28a(+) expression vec-
tor (Novagen, Madison, WI, USA). The amplified product
was then subcloned into respective restriction sites of pET-

In vitro conversion of the purified and refolded rproErv-
C
+CT
into its mature and active form was performed by
optimizing several parameters like proper activator (reduc-
ing agent), concentration of the activator, pH, temperature
and time. At a designated time interval, aliquots of the
sample were collected from the reaction mixture and mixed
with an equal volume of 2 · SDS ⁄ PAGE gel loading sam-
ple buffer containing 2–3 m
M irreversible inhibitor E-64
and analyzed by SDS ⁄ PAGE gel to optimize the time
required for complete maturation. Proenzyme processing of
rproErv-C
+CT
was compared with rproErv-C
DCT
by
SDS ⁄ PAGE analysis. Purity of the mature forms of rpro-
Erv-C
+CT
and rproErv-C
DCT
was established by western
blot analysis using rabbit antiserum raised against pure and
mature Erv-C from the plant latex as primary antibody
(Bangalore Genei, Bangalore, India) using the protocol
described earlier [29]. Wild-type Erv-C from plant latex was
also used for comparison.
Effect of external proteases on C-terminal processing

digestion by papain was carried out at pH 6.5 and 60 °C
for 0.5 h with 20 m
M cysteine, 2 mM EDTA and blocked
by E-64; trypsinization was carried out at pH 8.0 and
37 °C for 2 h and blocked by complete mini EDTA free
cocktail inhibitor (Roche, Mannheim, Germany); digestion
by chymotrypsin was carried out at pH 8.0 at 50 °C for 1 h
with 2 m
M CaCl
2
and blocked by complete mini EDTA free
cocktail inhibitor; pepsinization was carried out at pH 4.0
and 37 °C for 2 h and blocked by E-64 and pepstatin. Puri-
fied rproErv-C
+CT
was also digested using the abovemen-
tioned enzymes separately at 20 °C for 24 h under the same
conditions. The proteolytic digestion in each experiment
was checked by SDS ⁄ PAGE analysis.
Measurement of proteolytic activity
Substrate gel zymography using 0.1% gelatin as a substrate
was used to demonstrate the protease activity of recombi-
nant refolded proteases (rproErv-C
DCT
and rproErv-C
+CT
)
using a protocol described previously [29].
The specific activity of the recombinant proteases rpro-
Erv-C

Determination of optimum temperature for
enzymatic activity of rproErv-C
DCT
and
rproErv-C
+CT
T
opt
or ‘temperature optima’ is the temperature at which
the enzyme shows maximum activity. Proteolytic activity of
both the recombinant proteins (rproErv-C
DCT
and rproErv-
C
+CT
) was measured in the range 30–90 at 5 °C intervals
to determine the optimum temperature of activity (T
opt
) for
both recombinant proteins. The pro-proteases were first
converted to mature enzymes and the proteolytic activity
was then measured using azocasein as a substrate as
described above at specific temperatures.
Kinetic measurements using a chromogenic
peptide
The mature form of rproErv-C
+CT
, activated from 1 lM
proenzyme rproErv-C
+CT

where [E]
T
is the total concentration
of the active enzyme, the values of which were mea-
sured by active-site titration with the irreversible inhibitor,
E-64 using the above mentioned pNA containing sub-
strate, as described below. An extinction coefficient
of 8800
M
)1
Æcm
)1
at 410 nm for pNA was used for the
calculations.
Measurement of IC
50
value of E-64
Aliquots of 1 lM proenzyme (rproErv-C
+CT
and rproErv-
C
DCT
) were converted into their respective mature forms.
The irreversible inhibitor, E-64 was added in increasing
concentrations to the aliquots until the residual activity
reached 0. The residual activity (DA
410 nm
Æmin
)1
) was deter-

50
was expressed as the temperature of incubation at
which the enzyme showed 50% of maximum activity. The
half-life (t
1 ⁄ 2
) of an enzyme is the time it takes for the
residual activity to reduce to half of the original activity
after the enzyme is incubated at a particular temperature,
generally at T
max
. The t
1 ⁄ 2
values of two enzymes were esti-
mated by incubating the proteins at 65 °C(T
max
for both
the enzymes) for periods between 0 min and 8 h and then
the residual enzymatic activity was determined by azocasein
assay at 65 °C.
Molecular modeling of the mature Erv-C with
CT-extension (rmErv-C
+CT
)
The X-ray structure of mature domain of Erv-C, without
the CT-extension, was solved previously in our laboratory
(Protein Data Bank ID
2PNS). We have now performed a
BLASTP (http://blast.ncbi.nlm.nih.gov/) protein sequence
search of the amino acid sequence of CT-extension part
using RCSB Protein Data Bank (http://www.rcsb.org/pdb)

proteases bleomycin hydrolases (Protein Data Bank IDs
2CB5 and 1GCB) with the unprimed subsites of their cog-
nate enzymes were also considered during this manual
docking. The docked tetrapeptide fragment of the CT-
extension was used as an anchor position and the next
stretch of 19–11 residues, which showed a helical nature
from both the template conformation (Protein Data Bank
ID
1PV8, residues 320–329 of chain B) and the secondary
structure prediction using the program
DSC (Discrimination
of Protein Secondary Structure Class) [31] implemented in
DISCOVERY STUDIO 2.5 (Accelrys Inc., San Diego, CA,
USA), were positioned and fitted, taking into account
charge compatibility and hydrophobic interactions with
respect to the structure of mature Erv-C. The template
(Protein Data Bank ID
2X7X, residues 44–50 of chain A)
of the first seven residues of the CT-extension is a connect-
ing polypeptide between a b sheet and an a helix. In the
structure of the mature domain of Erv-C (Protein Data
Bank ID
2PNS) the C-terminus ends with a b sheet and
therefore we connected this seven-residue fragment of the
CT-extension to this b sheet of Erv-C here also, and ori-
ented it towards the previously fitted helical stretch (19–11).
Finally, these three fragments were joined by the connecting
sequences (residues 8–10 and 20) and the rotamer confor-
mation of the side chain of each residue was optimized.
The resulting complex of Erv-C together with the CT-exten-

structural interpretations in this study. In the model thus
generated, the length of the helix of CT-extension was
Role of C-terminal extension in a cysteine protease S. Dutta et al.
3022 FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS
reduced (residues 12–16) from the starting structure (resi-
dues 11–19). At the end of the simulation, the CT-extension
was isolated by deleting the mature domain and the exten-
sion was allowed to relax in a same simulation protocol as
described above for comparison. The simulation trajectory
was analyzed by ‘Analysis’ protocol of
DISCOVERY STUDIO
2.5.
The geometrical and structural consistencies of the model
structure of Erv-C together with CT-extension were evalu-
ated by different approaches. The stereochemical quality of
the model was evaluated using
PROCHECK [32] and VERIFY3D
[33]. This structure was further validated using web-based
software
QMEAN [34], PROQ [35] and SOLX [36]. All these val-
idations (Table S2) signify that the model is of good quality
and is reliable.
Acknowledgement
The work was partially supported by CSIR (Grant
No. 21 ⁄ 0653 ⁄ 06 ⁄ EMR-II), Govt. of India.
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Supporting information
The following supplementary material is available:
Fig. S1. SDS ⁄ PAGE showing C-terminal processing of
rproErv-C
+CT
by external proteases.
Fig. S2. The mature Erv-C with CT-extension.
Table S1. Yield of rproErv-C
+CT
and rproErv-C
DCT
from 1 L culture at different purification steps from
inclusion bodies.
Table S2.
CLUSTALW alignment and structural valida-
tions of the model structure of CT-extension.
Table S3. Raw data sets related to thermal stability
experiments.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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from supporting information (other than missing files)
should be addressed to the authors.
Role of C-terminal extension in a cysteine protease S. Dutta et al.
3024 FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS