Regular paper in the field of Biotechnology and topic of Gene and Protein Engineering

Efficient Expression and Purification of Recombinant Human m-calpain Using an
Escherichia coli Expression System at Low Temperature(Running title: Efficient purification of recombinant human m-calpain.)

Shoji Hata, Mika Ueno, Fujiko Kitamura, and Hiroyuki Sorimachi
Calpain Project, Department of Advanced Science for Biomolecules, Tokyo Metropolitan
Institute of Medical Science, 2-1-6 Kami-kitazawa, Setagaya-ku, Tokyo 156-8506, Japan.

Corresponding author: Shoji Hata, Ph.D.
Calpain Project, Department of Advanced Science for Biomolecules, Tokyo Metropolitan
Institute of Medical Science, 2-1-6 Kami-kitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
TEL: +81-3-5316-3124; FAX: +81-3-5316-3163.
E-mail: hata-sj@igakuken.or.jp.

1Summary

Calpain belongs to the superfamily of Ca2+-regulated cysteine proteases, which are
indispensable to the regulation of various cellular functions. Of the 15 mammalian calpain
isoforms, - and m-calpains are the best characterized. Both - and m calpain are ubiquitously
expressed and exist as heterodimers, containing a distinct 80-kDa catalytic subunit (CAPN1 and
CAPN2, respectively) and the common, 30-kDa regulatory subunit (CAPNS1). To date, various
expression systems have been developed for producing recombinant calpains for use instructural and physiological studies, however Escherichia coli systems have provenincompatible with large scale preparation of calpain, with the exception of rat m-calpain. Here,
we have established a highly efficient method to purify active recombinant human m-calpain
using an E. coli expression system at low temperature (22°C). This was achieved byco-expressing CAPN2 with a C-terminal histidine-tag, and CAPNS1, lacking the first
Gly-repeated region at the N-terminal. After 3 sequential passes through a chromatographic
column, approximately 5 mg of human m-calpain was homogenously purified from 1liter of E.
coli culture. Proteins were stable for several months. This is the first report of efficient,large-scale purification of recombinant human m-calpain using an E. coli expression system.

Keywords
bacterial expression, Gly-rich region, high yield, human m-calpain, large-scale purification2

Introduction

Calpain (Clan CA-C2, EC 3.4.22.17) belongs to a family of intracellular Ca2+-regulated cysteine
proteases, whose members share homology in the protease domain in almost all eukaryotes anda few bacteria. In the human genome, there are 15 calpain genes with ubiquitous or
tissue-specific expression. Calpains play pivotal roles in biological processes, including the cell
cycle, apoptosis, and myoblast fusion, through the limited proteolytic cleavage of diverse
substrates, although their physiological functions remain obscure. A number of pathologicalconditions, including muscular dystrophies, lissencephaly, cataracts, neurodegenerative
disorders, and gastric ulcers have been associated with aberrant calpain action (1, 2).
Two representative mammalian calpains, - and m-calpain, are ubiquitously expressed and havebeen extensively characterized. Both - and m- calpain exist as heterodimers, comprising thedistinct 80-kDa catalytic subunits CAPN1 and CAPN2, respectively, and a common 30-kDa
regulatory subunit, CAPNS1, and require M and mM levels of Ca2+, respectively, for theiractivation in vitro. CAPN1 and CAPN2 consist of a regulatory N-terminal anchor helix,
followed by a protease CysPc domain, a C2-like Ca2+/phospholipid-binding C2L domain and apenta-EF-hand PEF domain. CAPNS1 comprises a structurally flexible, Gly-rich GR domain
containing two Gly-repeated regions, and a PEF domain (Fig. 1). The PEF domains of thecatalytic and regulatory subunits interact with each other through their C-terminal EF-handmotifs, forming a heterodimer. In the absence of Ca2+, - and m-calpains are catalyticallyinactive, because the CysPC domain is separated into two protease core domains, PC1 and PC2,
preventing active site formation. The binding of Ca2+ to the PC1, PC2, C2L, and PEF domains,
induces structural changes that allow the domains to form a single protease core, involving
subunit dissociation and/or N-terminal autolysis in both subunits including degradation within3the GR domain (3-6).
Large-scale systems in E. coli or insect cells have been used to purify full-length or truncated,recombinant - and m-calpain for structural-functional studies, involving 3D analyses. However,a persistent problem has been that recombinant full-length calpains, including - and m-calpain,
aggregate as insoluble inclusion bodies within E.coli cells. In terms of large-scale purification ofrecombinant full-length calpain, using an E. coli expression system, this method has only beensuccessful for rat m-calpain, by co-expression of CAPN2 and CAPNS1 lacking the GR domain
at 30oC using compatible expression vectors (7, 8). Recombinant human m-calpain can beextracted as a soluble protein using insect cells, but it accumulates as an insoluble inclusion
body in E. coli cells (9). In this study, we have established a method for high-level expression
and purification of human m-calpain in a soluble and active form, using an E. coli system at a
low temperature.

4

Materials and methods

Reagents and antibodies
Chemical reagents were purchased from Sigma Inc. (MO, USA), Wako Pure chemicals Inc.
(Osaka, Japan), Kanto chemicals Inc. (Tokyo, Japan), and Nakarai Tesque Inc. (Kyoto, Japan).
Anti-His rabbit polyclonal antibody and anti-CAPNS1 mouse monoclonal antibody (clone P1)
were purchased from Santa Cruz Biotechnology Inc. (CA, USA) and American Type Culture
Collection (VA, USA), respectively.

Expression vectors
The cDNAs encoding human CAPN2, CAPNS1, CAPNS1G1, and CAPNS1G2 (forstructures, see Fig. 1) were amplified by PCR using pfu turbo polymerase (Agilent Technologies
Inc., CA, USA), and the sequences were verified after subcloning into compatible vectors forbacterial co-expression. Human CAPN2 was ligated into the pET24b(+) vector (Novagen,Darmstadt, Germany) to produce proteins fused with a C-terminal His-tag (CAPN2/pET), andhuman sequences CAPNS1, CAPNS1G1, or CAPNS1G2 were ligated into the pACpET24
vector (7) to produce proteins without peptide tags (CAPNS1/pACpET, CAPNS1G1/pACpET,or CAPNS1G2/pACpET, respectively). The pACpET24 vector was constructed by replacing
an 849-bp BamHI-HindIII fragment, excised from the pACYC177 vector (New England
Biolabs. Inc., MA, USA) with a 170-bp, BglII-HindIII fragment from the pET24b(+) vector.
The pACpET24 vector contains an ampicillin-resistant gene, the T7 promoter, and the A15
replication origin.

5Protein expression and purification
CAPN2/pET was co-transformed into E. coli BL21(DE3) (Novagen) with CAPNS1/pACpET,CAPNS1G1/pACpET, or CAPNS1G2/pACpET, and the transformants were stored as frozen
glycerol stocks at -80°C. For protein expression and purification, frozen transformants wereprecultured at 27°C overnight, in 30 ml of LB medium containing 100 g/ml ampicillin and 50
g/ml kanamycin (LB+Amp+Kn), and then cultures were diluted into 1 litter of LB+Amp+Kn,
and grown at 27°C with vigorous shaking until A600 reached 0.8–1.0. Protein expression was
induced by adding 0.2 mM isopropyl-thiogalactoside (IPTG), for 6 h at 22°C, with vigorous
shaking.
Purification was performed at 4°C unless otherwise indicated. Harvested cells were washedonce with ice-cold phosphate buffered saline, resuspended in 50 ml of ice-cold TED buffer (20mM Tris-Cl (pH 7.5), 1 mM EDTA, and 1 mM dithiothreitol (DTT)) containing 0.3 mM PMSF,
and lysed with a French Press (Ohtake Works, Tokyo, Japan). The cell lysate wasultracentrifuged at 55,000g for 30 min, and the recovered supernatant was filtered through a0.22 m-pore filter. The supernatant was applied to a DEAE-Toyopearl (Toyosoda Inc.,
Yamaguchi, Japan) anion exchange column (26 mm diameter, 124 mm length) equilibrated with
TED buffer. The column was washed with TED buffer and the protein eluted with a linear
gradient of 0–0.4 M NaCl in TED buffer, in 10 column volumes. Active fractions (total of 25ml) were pooled, and 5 M NaCl and 1 M MgCl2 added to final concentrations of 0.4 M and 5
mM, respectively, and filtered as described above. The pooled fractions were applied to a
HisTrap HP (16 mm diameter, 25 mm length) affinity column (GE healthcare Inc.,
Buckinghamshire, England) equilibrated with buffer A (20 mM Tris-Cl (pH 7.5), 0.4 M NaCl,and 1 mM DTT) containing 4 mM imidazole. The column was washed with 10 column volumesof the same buffer, and protein was eluted with a linear gradient of 4–250 mM imidazole in6buffer A, in 10 column volumes into collection tubes containing EDTA, to a final concentrationof 1 mM. Active fractions (total of 15 ml) were immediately dialyzed against TED buffer
overnight, pooled, and centrifuged at 20,000g for 20 min to remove debris. The dialyzed sample
was applied to a MonoQ HR10/10 anion-exchange column (GE healthcare), equilibrated with
TED buffer, and after washing the column with TED buffer, the protein was eluted with a linear
gradient of 0–0.55 M NaCl in TED buffer, in 5 column volumes. The purity of protein wasverified by SDS-PAGE. The peak fractions (1 ml/fraction) were stored at 4°C until use. For gel
filtration analysis, 0.7 g of the final peak fractions were applied to a HiLoad 16/10 Superdex
200 column (GE healthcare) equilibrated with TED buffer containing 150 mM NaCl.

Activity assay for calpain
The proteolytic activity of calpain was measured using casein (Sigma), as previously described,
with some modifications (10). Aliquots of the fractions eluted from columns were incubated at
30°C for 20 min, in reaction buffer (100 mM Tris-Cl (pH 7.5), 3 mg/ml casein, and 20 mM
2-mercaptoethanol) with 5 mM CaCl2 or 5 mM EDTA, in total volume of 50 l. Afterincubation, the reactions were stopped by the addition of 150 l of 7% (v/v) trichloroacetic acid,incubated on ice for 30 min, and centrifuged at 15,000 rpm for 15 min at 4°C. A280 of thesupernatant was measured using a spectrophotometer (SmartSpec-3000, Bio-Rad Laboratories
Inc., CA, USA). One unit of calpain caseinolytic activity was defined as a Ca2+-dependent
increase in A280 of 1.0, in 1-hour, in a 1-ml scale reaction. The proteolytic activity was alsomeasured using a fluorogenic substrate, succynyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide(Suc-LLVY-MCA) (Peptide Institute Inc., Osaka, Japan), with some modifications (11). The
final peak fraction was incubated with 10–500 mM substrate for 10 min at 30°C, in 100 mM
Tris-Cl (pH 7.5), 5 mM CaCl2, 20 mM -mercaptoethanol, and 0.2%7

3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic acid (CHAPS) in a total volume of
100 l. Reaction was stopped by adding 100 l of 10% SDS and 1.2 ml of 0.1 M Tris-Cl (pH9.0). Release of 7-amino-4-methylcoumarin was monitored by a spectrofluorophotometer(RF-1500, Shimadzu, Osaka, Japan), with excitation and emission wavelengths at 380 nm and
460 nm, respectively. The kinetic parameters (Km and Kcat) were calculated from
Lineweaver-Burk plots.

Casein zymography
Casein zymography was performed as previously described (12). Two g of the final peakfraction, 1 g of recombinant human m-calpain purified by Sf9/vaculovirus expression system,and 25 g of non-transfected COS7 cell lysate were added to Native-PAGE loading buffer (250mM Tris-Cl, pH 6.8, 50% glycerol, 1 mM DTT, and 0.005% bromophenol blue), and run on a
12% polyacrylamide gel containing 2mg/ml casein at 125 V for 3 h. After electrophoresis, thegel was incubated overnight in Ca2+ incubation buffer (50 mM Tris-Cl, pH 7.0, 5 mM CaCl2,
and 10 mM DTT), stained with Coomassie Brilliant Blue (CBB) R250, and destained withdestain solution (10% acetic acid and 30% methanol).

SDS-PAGE and western blotting
SDS-PAGE was performed as previously described (13). Samples were added to SDS-PAGE
loading buffer (62 mM Tris-Cl (pH 6.8), 143 mM 2-mercaptoethanol, 2% SDS, 0.005%
bromophenol blue, and 10% glycerol), boiled for 5 min, and analyzed on 10 or 12%
polyacrylamide gels. The gels were stained with CBB R250. For western blotting, proteins were
subjected to SDS-PAGE, transferred to PVDF membranes (Immobilon-P, Millipore Inc., MA,
USA), and probed with appropriate antibodies. Signals were detected using a POD immunostain

8

kit (Wako Pure Chemicals Inc.).

9

Results and Discussion

Expression of recombinant human m-calpain
It was previously reported that fully active, recombinant rat m-calpain was successfully
produced at 30oC by bacterial co-expression of CAPN2 with a C-terminal His-tag, and 21K,
CAPNS1 lacking the N-terminal GR domain (amino acid (aa) 1–86) and using the twocompatible expression vectors, pET24b(+) and pACpET24, with distinct antibiotic-resistantgenes, and replicons (7, 8). We applied this strategy to human m-calpain, although it wasreported that, in E. coli cells, recombinant human m-calpain formed inclusion bodies that could
not be renatured (9).
When CAPN2/pET and CAPNS1/pACpET were used in bacterial co-expression, expression
levels of CAPNS1 were very low, and the CAPN2 protein was almost insoluble under the tested
conditions (Figs. 2A and 2B). After DEAE column chromatography, the soluble lysate gave
CAPN2-enriched, but not active fractions (data not shown). This suggests that the two Gly
repeat regions in the GR domain caused a deleterious interaction with the host’s
post-translational machinery, as in the case of recombinant rat m-calpain containing full-length
CAPNS1 (8). Therefore, instead of a CAPNS1/pACpET construct, we used truncated forms of
CAPNS1, CAPNS1G1/pACpET and CAPNS1G2/pACpET. As shown in Fig. 1,
CAPNS1G1/pACpET lacks one Gly repeat (aa 1–25), while CAPNS1G2/pACpET lacks both
Gly repeats (aa 1–58), in the GR domain. As a result, the yields and solubility of not only
CAPNS1G1 and CAPNS1G2 but also CAPN2 showed similar improvement at a lower
growth temperature (22°C), after adding IPTG. In addition, the C-terminal His-tag fused to
CAPN2 was not truncated during protein expression (Figs. 2A and 2B). We therefore
co-expressed CAPNS1G1, the closest to native form, with CAPN2, for large-scale

10

purification.

Purification of recombinant human m-calpain
As described in Material and Methods, purification of co-expressed CAPN2+CAPNS1G1 was
performed by sequential column chromatography, using DEAE-Toyopearl, HisTrap, andMonoQ columns. At each column step, the eluted fractions were monitored for calpain activity
by caseinolytic assay. Calpain activity was detected in the fractions containing 200–250 mM
NaCl from the DEAE column, 50–125 mM imidazole from the HisTrap column, and 450 mM
NaCl from the MonoQ column. SDS-PAGE analysis of the peak active fractions eluted fromeach column, indicates that CAPN2 and CAPNS1G1 were co-eluted and almost
homogeneously purified (Fig. 2C). The final preparation gave a clear band at the same position
as a control sample in casein zymography (Fig. 2D). Gel filtration analysis of the final
preparation showed a single elution peak at a position estimated to be 110 kDa, which is almost
identical to theoretical total molecular weight of CAPN2 with the His-tag and CAPNS1G1(Fig. 3). These results indicate that CAPN2 and CAPNS1G1 formed a heterodimer withouthigh molecular weight aggregates or dissociated molecules. The results of purification aresummarized in Table 1. From 1 liter of E. coli culture, we obtained 5.8 mg of purified
recombinant human m-calpain, with a yield of 51.1%, which was higher than that ofrecombinant human m-calpain obtained from a Sf9/baculovirus expression system (9). This
protein was stable for several months. The final preparation showed a half maximal activity of
approximately 0.5 mM Ca2+, and a specific activity of 334 U/mg for casein, and Km and Kcat
values for Suc-LLVY-MCA were determined as 0.322 mM and 0.066 sec-1, respectively (Fig. 4).
These values are similar to those reported previously for recombinant or native m-calpain (9-11,14), indicating that the N-terminal 25 amino acid truncation of CAPNS1 did not affect11

enzymatic properties of intact calpain, such as heterodimerization, specific activity, and theCa2+-requirement.
In conclusion, we have established a highly efficient bacterial expression and purification
system for recombinant human m-calpain, a system that is easier to manipulate than theSf9/baculovirus expression system. Our methodology has the potential to assist researchers withstructural and physiological studies. Sustained activation of - and m-calpains is associated withvarious pathological states, making these proteases an obvious potential therapeutic target to
reduce or prevent a spectrum of human diseases. Great efforts are underway to develop selective
calpain inhibitors, and the use of human calpains rather than mammalian orthologues would beof great benefit in this process. In addition, our expression system and constructs may provide
resources for further analysis into the physiological significance of the hydrophobicity exhibitedby Gly-repeats in the calpain GR domain.

12

Acknowledgements
We thank all laboratory members for their technical support.

13

References

1. Goll, D. E., Thompson, V. F., Li, H., Wei, W. and Cong, J. (2003) The calpain system. Physiol. Rev. 83,
731-801
2. Sorimachi, H., Hata, S. and Ono, Y. (2011) Impact of genetic insights into calpain biology. J Biochem
150, 23-37
3. Strobl, S., Fernandez-Catalan, C., Braun, M., Huber, R., Masumoto, H., Nakagawa, K., Irie, A.,
Sorimachi, H., Bourenkow, G., Bartunik, H., Suzuki, K. and Bode, W. (2000) The crystal structure ofcalcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium.
Proc. Natl. Acad. Sci. USA. 97, 588-592
4. Hosfield, C. M., Elce, J. S., Davies, P. L. and Jia, Z. (1999) Crystal structure of calpain reveals the
structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation. EMBO J.
18, 6880-6889
5. Moldoveanu, T., Gehring, K. and Green, D. R. (2008) Concerted multi-pronged attack by calpastatin to
occlude the catalytic cleft of heterodimeric calpains. Nature 456, 404-408
6. Moldoveanu, T., Hosfield, C. M., Lim, D., Elce, J. S., Jia, Z. and Davies, P. L. (2002) A Ca2+ switchaligns the active site of calpain. Cell 108, 649-660
7. Elce, J. S., Hegadorn, C., Gauthier, S., Vince, J. W. and Davies, P. L. (1995) Recombinant calpain II:
improved expression systems and production of a C105A active-site mutant for crystallography. ProteinEng. 8, 843-848
8. Elce, J. S. (2000) Expression of m-calpain in Escherichia coli. Methods Mol. Biol. 144, 47-54
9. Masumoto, H., Yoshizawa, T., Sorimachi, H., Nishino, T., Ishiura, S. and Suzuki, K. (1998)
Overexpression, purification, and characterization of human m-calpain and its active site mutant,
m-C105S-calpain, using a baculovirus expression system. J. Biochem. 124, 957-961
10. Ishiura, S., Murofushi, H., Suzuki, K. and Imahori, K. (1978) Studies of a calcium-activated neutral
protease from chicken skeletal muscle. I. Purification and characterization. J. Biochem. 84, 225-230
11. Sasaki, T., Kikuchi, T., Yumoto, N., Yoshimura, N. and Murachi, T. (1984) Comparative specificity
and kinetic studies on porcine calpain I and calpain II with naturally occurring peptides and synthetic
fluorogenic substrates. J Biol Chem 259, 12489-12494
12. Arthur, J. S. and Mykles, D. L. (2000) Calpain zymography with casein or fluorescein isothiocyanate
casein. Methods Mol Biol 144, 109-116
13. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head ofbacteriophage T4. Nature 227, 680-685
14. Inomata, M., Nomoto, M., Hayashi, M., Nakamura, M., Imahori, K. and Kawashima, S. (1984)
Comparison of low and high calcium requiring forms of the calcium-activated neutral protease (CANP)

14

from rabbit skeletal muscle. J. Biochem. 95, 1661-1670

15

Table 1. Purification of recombinant human m-calpain
Volume(ml)

Total
proteins(mg)

Total activity Specific activity(U, x 103)

(U/mg)

Yield(%)

Purification
ratio(fold)

Total

50.0

625

3.78

6.00

100

1.00

Sup

45.0

464

3.51

8.00

92.9

1.30

DEAE

25.0

42.5

3.47

82.0

91.7

13.6

HisTrap

16.4

13.8

2.30

166

60.8

27.7

MonoQ

3.00

5.80

1.93

334

51.1

55.6

Sup, soluble lysate.

16

Figure legends

Fig.1 Schematic illustration of the human CAPN2, CAPNS1, CAPNS1G1, and
CAPNS1G2 constructs used in this study.

Fig. 2 Expression and purification of recombinant human m-calpain. CAPN2 wasco-expressed with CAPNS1, CAPNS1G1, or CAPNS1G2 at 30°C (A) or 22°C (B). Each E.
coli sample was lysed, fractionated, and subjected to SDS-PAGE (CBB staining, left panels) andwestern blot analysis (right panels). (C) CAPN2 and CAPNS1G1 were co-purified bysequential column chromatography, using DEAE-Toyopearl, HisTrap, and MonoQ columns.
Active peak fractions at each purification step were analyzed by SDS-PAGE. M, protein marker;
T, total lysate; S, soluble lysate; P, insoluble fraction; DE, His, and MQ, active peak fractions
from the DEAE-Toyopearl, HisTrap, and MonoQ columns, respectively. (D) Casein
zymography of the peak fraction at MonoQ column. Two g of the peak fraction (lane 2) was
subjected to casein zymography as described in Material and Methods with 1 g of recombinanthuman m-calpain purified by Sf9/vaculovirus expression system (lane 1) and 25 g ofnon-transfected COS7 cell lysate (lane 3) as controls.

Fig. 3 Elution profile of purified CAPN2 and CAPNS1G1 from a HiLoad 16/10 Superdex
200 gel filtration column. (left) The solid line represents A280 of the column eluate. (right)
Molecular weight determination of the peak eluate. Values of partition coefficient (Kav) were
calculated, and the Kav values of marker proteins show linear correlation with the logarithmicvalues of their molecular weights. Molecular weight of the peak eluate was estimated to be 110
kDa from the regression line, which was almost identical to the theoretical total molecular17

weight of CAPN2 with the His-tag and CAPNS1G1 (108 kDa).

Fig. 4 Ca2+-requirement of the purified recombinant human m-calpain. Purified
recombinant human m-calpain was incubated at 30°C in the presence of varying concentrationsof Ca2+ as described in the Materials and Methods. Activity was standardized by defining thevalue of pCa=2.5 as 100%. The Ca2+ concentration for half maximal activity was approximately
0.5 mM.

18

Funding
This work was supported by MEXT.KAKENHI 18076007 (to H.S.), 20780106 and 23780152(to S.H.), a Kato Memorial Bioscience Foundation research grant (to S.H.), JSPS.KAKENHI
20370055 (to H.S.), and a Takeda Science Foundation research grant (to H.S.).

Conflict of interest
The authors declare no conflict of interest relating to this work.

19

Hata, S. et al., Figure 1

CAPN2

PEF

CysPc

(aa) 1

N

PC1

PC2

C

H N

700

C2L

(His-tag)
GKLAAALEHHHHHH

EF-hand motif
CAPNS1(aa) 1CAPNS1∆G1CAPNS1∆G2

GR

98

PEF

268

Gly Gly(aa) 2698268(aa) 59 98

268

Gly

Hata, S. et al., Figure 2
A(CBB stain)(western blot)

CAPN2-C-His

empty
vectors
(kDa)

T

S

+
CAPNS1

P

T

S

+
+
CAPNS1∆G1 CAPNS1∆G2

P

T

S

P

T

S

CAPN2-C-His

empty
vectors

P

175

T

83

S

+
CAPNS1

P

T

S

+
+
CAPNS1∆G1 CAPNS1∆G2

P

T

S

P

T

S

P

CAPN2-C-His

62

anti-His

47
32
25

CAPNS1CAPNS1∆G1CAPNS1∆G2

anti-CAPNS1

16

B(CBB stain)(western blot)

CAPN2-C-His

empty
vectors
(kDa)

175
83
62

T

S

+
CAPNS1

P

T

S

+
+
CAPNS1∆G1 CAPNS1∆G2

P

T

S

P

T

S

CAPN2-C-His

empty
vectorsP

T

S+
CAPNS1

P

T

S

+
+
CAPNS1∆G1 CAPNS1∆G2

P

T

S

P

T

S

P

CAPN2-C-His

anti-His

47
32
25

CAPNS1CAPNS1∆G1CAPNS1∆G2

16

anti-CAPNS1

Hata, S. et al., Figure 2 (continued)
C
(kDa)
175

M T

S DE His MQ

83

CAPN2-C-His

62
47
32
25

CAPNS1∆G1

16

D

1

2

3
µ-calpain
m-calpain

Hata, S. et al., Figure 3

0.7

0.6

0.6
0.5
A280

catalase(206 kDa)

0.4

BSA ovalbumin(67 kDa) (43 kDa)

0.3
0.2

0.2

0.1

0.1
5

10
elution (ml)

15

BSA

0.4
Kav 0.30

ovalbumin

0.5(fr. No.)0

purified sample
catalase

1

5

10

Molecular weight (Da, x 104)20

30

Hata, S. et al., Figure 4

80
60
40
20

∞

7

6

5 4.5 4 3.5 3 2.5 2

pCa

0

Relative activity (%)

100