Protein Expression and Purication 31 (2003) 231–239
www.elsevier.com/locate/yprep
Expression and purication of the h1 and h2 isoforms of calponin
Jian-Ping Jin,a,b,* Di Wu,a,b Jimin Gao,b Rita Nigam,b and Stephen Kwongb
a Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4970, USA
b Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1
Received 25 March 2003, and in revised form 14 May 2003
Abstract
Three homologous calponin isoforms, named h1, h2, and acidic calponins, have been found in birds and mammals. Based
primarily on studies of chicken gizzard smooth muscle (h1) calponin, calponin has been identied as a family of actin-associated
proteins that inhibit actomyosin ATPase activity. Evolutionary divergence of the calponin isoforms suggests dierentiated function.
While the role of h1 calponin in smooth muscle contraction is under investigation, h2 calponin has been shown regulating the
function of actin cytoskeleton. Using cloned cDNA, we expressed mammalian h1 and h2 calponins in Escherichia coli. We have
developed eective methods to purify biologically active h1 and h2 calponin proteins from transformed bacterial culture. The
puried calponin isoform proteins were used to generate monoclonal antibodies that reveal epitopic structure dierence between h1
and h2 calponins. Together with their dierential expression in tissues and during development, the structural diversity of h1 and h2
calponins suggests non-redundant physiological function. Nevertheless, h1 and h2 calponins bind F-actin with similar anity,
indicating a conserved mechanism for their role in regulating actin laments in smooth muscle and non-muscle cells.
2003 Elsevier Science (USA). All rights reserved.
Calponin is a family of actin lament-associated lacking calponin is contractile upon physiological sim-
proteins found in smooth muscle and non-muscle cells. ulation, the expression of h1 calponin is correlated to
Encoded by homologous genes, three calponin isoforms, the sensitivity to norepinephrine activation [19]. The
h1 (basic) [1,2], h2 (neutral) [3], and acidic [4,5], have hypothesis that h1 calponin is a modulatory protein in
been identied. The physiological and pathological sig- smooth muscle contraction has been demonstrated fur-
nicance of the dierent calponin isoforms is largely ther in gene knockout mice, in which the lack of h1
unknown. h1 Calponin is the major calponin found in calponin in smooth muscle resulted in faster velocity of
smooth muscle and has been extensively analyzed in unloaded shortening [20,21]. While the physiological
many previous studies. In vitro protein binding studies function of h1 calponin in smooth muscle contraction
have demonstrated that calponin binds actin [6–8] and remains to be investigated, it has been shown that h1
tropomyosin [9,10], suggesting a role in the function of calponin is required for agonist-induced signal trans-
smooth muscle thin laments. h1 Calponin inhibits the duction in smooth muscle cells [22] and the survival of
actin-activated MgATPase activity of smooth muscle h1 calponin gene knockout mice may be based on
myosin [7,11–13], the movement of actin laments over compensation from changes in related proteins such as
immobilized myosin in in vitro motility assays [14,15], the level of actin [20].
and the force development and shortening velocity of The function of h2 calponin has not been extensively
skinned smooth muscle [16,17]. These data suggest that studied. Structural conservation suggests that the h1, h2,
in addition to the myosin-based regulation [18], h1 cal- and acidic calponins may function via similar molecular
ponin may play a modulatory role in smooth muscle mechanisms. On the other hand, the three calponin
contraction. Although rat vascular smooth muscle isoforms have signicantly diverged during vertebrate
evolution (Fig. 1). Although h1 and h2 calponins are
similar in size, their notable sequence diversity as dem-
* Corresponding author. Fax: 1-216-368-3952. onstrated by the dierent overall charges suggests that
E-mail address: jxj12@po.cwru.edu (J.-P. Jin). they have adapted to dierent biological processes. The
1046-5928/$ - see front matter 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S1046-5928(03)00185-2
232 J.-P. Jin et al. / Protein Expression and Purication 31 (2003) 231–239
indicating a conserved mechanism for their role in
regulating actin laments in smooth muscle and non-
muscle cells.
Materials and methods
Cloning of cDNAs encoding mouse h1 and h2 calponins by
reverse transcription-coupled polymerase chain reaction
We cloned a cDNA encoding mouse h1 calponin by
reverse transcription-coupled polymerase chain reaction
Fig. 1. Phylogenetic analysis of calponin isoforms. Amino acid se- (RT-PCR) from the uterus smooth muscle of pregnant
quences of human h1 (GenBank Accession No. S80560), h2 (D83735) 129SvJ mice. Total RNA was extracted from the fresh
and acidic (S80562) calponins, mouse h1 (Z19542) and h2 (Z19543) tissue with the TRIZOL reagent (Gibco-BRL) according
calponins, pig h1 (Z19538) and h2 (Z19539) calponins, rat h1 (X71071)
to manufacturers protocol. Using 2lg of the total RNA
and acidic (U06755) calponins, ferret h1 calponin (AF323674), and
chicken h1 calponin (M63559) were compared to construct a phylo- as template, an oligonucleotide primer complementary
genetic tree. The pI of each calponin isoform was calculated from to the sequence anking the translation stop codon of
amino acid sequences to demonstrate the dierences between the three mouse h1 calponin mRNA [3] was used for reverse
calponins. transcription of mouse h1 calponin cDNA. Double
stranded cDNA was then synthesized by PCR using this
expression of h1 calponin in smooth muscle is up-reg- primer paired with a primer corresponding to the se-
ulated during development, suggesting a function in quence anking the translation initiation codon of
mature myolaments [19,23–27]. In contrast, h2 calpo- mouse h1 calponin mRNA.
nin is expressed at higher levels in growing and remod- We cloned further a cDNA encoding mouse h2 cal-
eling smooth muscle tissues [27]. Expression of h2 ponin from RNA extracted from fresh stomach smooth
calponin was detected by RT-PCR in non-smooth muscle tissue of adult 129SvJ strain mouse. To amplify
muscle organs such as the heart [28]. We have recently the low abundant h2 calponin mRNA in stomach tissue,
shown high levels of h2 calponin expression in bro- a short oligo nucleotide primer complementary to a
blasts [29]. Over-expression of h1 [30] or h2 [27] calponin segment in the 30-untranslated region was used for the
inhibits the rate of cell proliferation, suggesting its role reverse transcription of rst strand cDNA. PCR was
in modulating actin-cytoskeleton during cytokinesis followed using a pair of primers corresponding to the
[27]. The potential role of calponin in non-contractile regions anking the translation initiation and termina-
function is supported by the observation that calponin tion codons, respectively, of the mouse h2 calponin
may participate in mitogen-activated protein kinase and mRNA [3].
protein kinase C signaling [31,32]. Restriction enzyme cloning sites were constructed
Comparative studies of h1 and h2 calponins can in the oligonucleotide primers. The double-stranded
provide useful information for understanding the phys- cDNA synthesized by PCR was digested with NdeI and
iological function of calponin. Biochemical studies using EcoRI and puried by the Prep-A-Gene glass bead-
puried protein form the foundation of functional binding method (Bio-Rad Laboratories) according to
characterization of calponin. Puried protein also pro- manufacturers protocol. The cDNA insert was ligated
vides standards for detecting calponin isoform expres- to NdeI–EcoRI-cut pAED4 prokaryotic expression
sion and materials for structural characterization. vector [34]. After transformation of JM109 strain of
Expression of cloned cDNA in bacteria is an eective Escherichia coli, ampicillin-resistant colonies containing
approach to prepare calponin isoform proteins. While the recombinant plasmids were identied by PCR for
the expression and purication of basic h1 calponin cDNA insert with appropriate sizes. The cloned h1 and
have been described in a procedure for chicken gizzard h2 calponin cDNAs were conrmed by DNA sequenc-
calponin [33], the method for expression and purica- ing using dideoxy chain termination method as de-
tion of h2 calponin has not been reported. In the present scribed previously [35].
study, we developed eective methods for the expression
and purication of mammalian h1 and h2 calponins Expression and purication of mouse h1 and h2 calponins
from cloned cDNA. The puried calponin isoforms
were used to develop specic monoclonal antibodies The procedures were carried out at 4 °C unless spec-
(mAbs) that reveal dierent epitopic structures in h1 ied.
and h2 calponins. Protein binding assays showed that The recombinant pAED4 expression plasmids were
h1 and h2 calponins bind F-actin with similar anity, used to transform BL21(DE3)pLysS strain of E. coli [36]
J.-P. Jin et al. / Protein Expression and Purication 31 (2003) 231–239 233
for the expression of mouse h1 or h2 calponin proteins. linear KCl gradient and the h1 calponin fractions were
Two times tryptone–yeast broth containing 100 lg/mL identied by SDS–PAGE, dialyzed, and concentrated by
ampicillin and 25 lg/mL chloramphenicol was inocu- lyophilization. The protein was then dissolved in a small
lated with a single colony of the freshly transformed volume of 6M urea, 0.2 M KCl, and 10mM imidazole–
bacteria and incubated at 37 °C with vigorous shaking. HCl, pH 7.0, and fractionated on a preparative Supe-
When the OD600 reached 0.4–0.5, the cultures were in- rose 12 gel ltration column for FPLC to remove the
duced with 0.4 mM isopropyl-1-thio-b-D-galactopyr- remaining contamination protein with a molecular
anoside (IPTG) and incubated further for 3h. The weight close to that of h1 calponin (Fig. 3). The column
bacteria were harvested by centrifugation, re-suspended fractions were examined by SDS–PAGE and those
in 50 mL of 50 mM Tris–HCl buer, pH 8.0, 2.5 mM containing pure h1 calponin were lyophilized after di-
EDTA, and lysed by three passes through a French alysis against 100 volumes of 0.5% formic acid for three
press at 1000 psi. The lysate was centrifuged to remove changes.
the insoluble materials and fractionated by step am- The h2 calponin in the bacterial lysate was enriched
monium sulfate precipitation. Predicted from the amino in the ammonium sulfate fraction of 0–30% saturation
acid sequences, the h1 and h2 isoforms of calponin have
signicantly dierent biochemical properties (Fig. 2).
Therefore, dierent procedures have been developed for
their purication.
As shown in Fig. 3, h1 calponin was present in the
ammonium sulfate fraction of 20–40% saturation. After
dialysis to remove the salt, urea was added to 6M and
the pH was adjusted to 7.0 in 20 mM imidazole–HCl
buer containing 0.1 mM EDTA and 6mM b-mercap-
toethanol for ion-exchange chromatography fraction-
ation of the basic h1 calponin (isoelectric point (pI) 9)
on a CM52 cation-exchange column. The protein solu-
tion was centrifuged to remove a small amount of pre-
cipitates and loaded onto the column equilibrated in 6M
urea, 0.1 mM EDTA, 6mM b-mercaptoethanol, and
10 mM imidazole–HCl, pH 7.0. After washing, the col-
umn-bound proteins were eluted with a 0–500 mM linear
Fig. 3. Bacterial expression and purication of mouse h1 and h2 cal-
KCl gradient in the same buer and the fractions were ponins. The recombinant expression plasmids encoding mouse h1 and
analyzed by SDS–polyacrylamide gel electrophoresis h2 calponins were used to transform BL21(DE3)pLysS E. coli. The
(SDS–PAGE). The protein peak containing h1 calponin transformed bacteria were cultured in liquid media at large scales and
was dialyzed to remove salt and re-fractionated on a induced with IPTG. The h1 and h2 calponins expressed in E. coli were
puried by serial fractionations using ammonium sulfate precipitation,
DE52 anion exchange column in 6M urea, 0.1 mM
ion-exchange, and gel ltration chromatographies. The SDS–PAGE
EDTA, 6mM b-mercaptoethanol, and 20 mM Tris– gels summarize the results of expression, each step of purication, and
HCl, pH 10. The column was eluted with a 0–300 mM nal products of the calponin isoform proteins.
Fig. 2. Predicted charge prole of h1 and h2 calponins. The calculated charge proles of mouse h1 and h2 calponins against dierent pH are shown.
The predicted charge property was used to direct designing ion-exchange chromatography purication of the basic and neutral calponin isoforms.
234 J.-P. Jin et al. / Protein Expression and Purication 31 (2003) 231–239
(Fig. 3). After dialysis to remove the salt, urea was were examined by SDS–PAGE and those containing
added to 6 M and the pH was adjusted to 8.8 in 10 mM pure h2 calponin (Fig. 3) were lyophilized after dialysis
Tris–HCl buer, 0.1 mM EDTA, and 6mM b-mercap- against 0.5% formic acid as above.
toethanol. After clarication by centrifugation, the The puried mouse h1 and h2 calponins were veried
protein solution containing the neutral h2 calponin by Western blotting using specic antibodies as previ-
(pI 7.6) was loaded on a DE52 anion exchange column ously described [19] and by amino acid analysis at the
for chromatography fractionation. After washing away Protein Sequencing Facility, University of Calgary.
the unbound proteins, the column was eluted with a 0–
300 mM linear KCl gradient in the same buer. The h2 Anti-calponin antibodies
calponin peak was identied by SDS–PAGE, dialyzed to
remove urea and salt, and lyophilized as above. To Two mouse mAbs (CP1 and CP3, IgG1j and Ig-
purify further the h2 calponin to homogeneity, the G2bj, respectively) previously developed by immuni-
preparation was fractionated further on a Sephadex G75 zation with puried chicken gizzard calponin [37] were
gel ltration column equilibrated in 6M urea, 0.2 M used in the present study. Both CP1 and CP3 cross-react
KCl, 6 mM b-mercaptoethanol, and 10 mM imidazole with mammalian h1 calponin but not h2 calponin (Figs.
buer, pH 7.0. Fractions from the gel ltration column 4 and 5).
The puried mouse h2 calponin protein was used as
immunogen for the preparation of mAbs against h2
calponin. The immunization of Balb/c mice and hy-
bridoma fusion were performed as described previously
[27,37]. The hybridomas were screened by enzyme-
linked immunosorbent assay (ELISA) against puried
h2 calponin using anti-mouse total immunoglobulin
second antibody for clones secreting anti-h2 calponin
mAb. Despite the weak immunogenicity of mouse h2
calponin in immunizing mice, we were able to obtain
signicant anti-h2 calponin antibody titer in the mouse
sera. Multiple hybridoma cell lines secreting high af-
nity anti-h2 calponin mAb were obtained, although
most of them produced IgM. The mAb specicity was
examined by Western blotting against puried h1 and
h2 calponins. Using Western blots transferred from
SDS–PAGE that resolves the size dierence between h1
and h2 calponins, the mAb specicity was veried on
Fig. 4. Dierential mobility of h1 and h2 calponins in SDS–PAGE and adult mouse urinary bladder tissue expressing both
specicity of anti-calponin isoform antibodies. The SDS–PAGE shows calponin isoforms [27]. An h2 calponin-specic mAb,
dierential mobility of mouse h1 (partially puried) and h2 calponins
CP21 (IgMj) [27], was used in the present study.
together with total protein homogenates of 3-day-old mouse urinary
bladder sample. The Western blots demonstrate the specicity of the The puried mouse h2 calponin was also used to
four anti-calponin antibodies used in this study. immunize a New Zealand White rabbit to produce
Fig. 5. Epitope analysis of h1 and h2 calponin isoforms. ELISA titration was carried out to compare the diversity as well as conservation between the
epitopic structure of h1 and h2 calponins. The gures show the titration curves for the anti-h1 mAbs CP1 and CP3 (A), the anti-h2 calponin mAb
CP21 (B), and the RAH2 rabbit antiserum (C). The results show the presence of h1 and h2 calponin-specic epitopes recognized by the mAbs.
Nevertheless, the titration curves of RAH2 polyclonal antibody against h1 and h2 calponins demonstrate the presence of shared epitopes in the two
calponin isoforms.
J.-P. Jin et al. / Protein Expression and Purication 31 (2003) 231–239 235
polyclonal antisera. The rabbit was injected intramus- plate at room temperature for 45 min followed by nal
cularly with 1 mg of the h2 calponin immunogen mixed washes and H2O2–ABTS substrate colour reaction.
in Freunds complete adjuvant and boosted three times The A405 nm of triplicate assay wells was monitored at a
with the same amounts of antigen in Freunds incom- series of time points using automated microplate
plete adjuvant at three-week intervals. Upon the detec- reader. The titration curves were plotted against the
tion of high serum antibody titer against h2 calponin, concentrations of calponin and the statistical signi-
the rabbit was sacriced to collect the immune serum cance of the dierences was examined by Students
(RAH2) [19]. t test.
ELISA epitope analysis Protein sequence analysis
We applied antibody epitope anity titration to ex- The charge proles of h1 and h2 calponins were
amine the structural diversity and conservation of h1 predicted using the Protean computer program
and h2 calponins. The epitope analysis was carried out (DNAStar). Phylogenetic analysis of calponin isoforms
by ELISA as described previously [38,39]. The puried and primary structure alignment for mouse h1 and h2
mouse h1 and h2 calponins were dissolved in Buer A calponins were done by the Clustal method using the
(0.1 M KCl, 10 mM piperazine-N,N0-bis(2-ethanesulf- MegAlign program (DNAStar).
onic acid) (PIPES), pH 7.0, and 3 mM MgCl2), at equal
concentrations for coating on microtiter plates. After
washing and blocking using phosphate-buered saline Results and discussion
(PBS) containing 0.05% Tween 20 (PBS-T), the plates
were incubated with serial dilutions of anti-calponin Evolutionary divergence of calponin isoforms
antibodies in PBS-T containing 0.1% bovine serum al-
bumin (BSA), followed by reactions with horseradish The phylogenetic tree in Fig. 1 demonstrates the
peroxidase (HRP)-conjugated anti-mouse total immu- evolutionary relationships among h1, h2, and acidic
noglobulin or anti-rabbit IgG second antibody and calponins in avian and mammalian species. The results
H2O2/2,20-azinobis-(3-ethylbenzthiazolinesulfonic acid show that the primary structure of each calponin iso-
(ABTS) substrate. The binding between the anti-calpo- form in birds and mammals is more conserved than
nin antibody and the calponin isoforms was quantied those of the three calponin isoforms in each species. The
by A405nm readings recorded at a series of time points by relationships indicate that the three calponin isoforms
an automated microplate reader (Benchmark, Bio-Rad). diverged early during vertebrate evolution. This phylo-
The A405nm readings from a time point within the linear genetic pattern is similar to that among the three tro-
range of the color development were used to construct ponin T (TnT) genes encoding cardiac, slow, and fast
the titration curves. The titrations were done in tripli- skeletal muscle TnT [40], suggesting a conserved func-
cate. tion of the calponin isoforms in dierent cell types. It is
interesting to note that the structure of h2 calponin
ELISA-based solid-phase actin-binding experiments (neutral) has diverged signicantly from that of h1 cal-
ponin (basic) and the acidic calponin may present an
ELISA-based protein-binding experiments [39] were intermediate isoform (Fig. 1). In other words, the
carried out to characterize the interactions of h1 and smooth muscle-specic h1 calponin is evolutionarily
h2 calponins with F-actin. Microtiter plates were more distant from h2 calponin than from acidic calpo-
coated at 4 °C overnight with smooth muscle F-actin nin. The structural dierence between h1 and h2 cal-
puried from chicken gizzard as described previously ponins may suggest specic adaptations to their activity
[39] (30 lg/ml and 0.1 ml/well) in Buer A as described and cellular environment. H1 calponin is the major
above. After three washes to remove the excess actin calponin found in smooth muscle [19,37] and has been
and block the remaining plastic surface with Buer A investigated as a potential modulator of smooth muscle
containing 0.05% Tween 20 (Buer T), the plates were contraction in many previous studies. In contrast, h2
incubated with serial dilutions of mouse h1 or h2 calponin is much less abundant in smooth muscle and
calponin in Buer T containing 0.1% BSA at room also expressed in certain non-muscle cells such as -
temperature for 2h. The plates were then washed three broblasts [29]. The level of h2 calponin expressed in
times with Buer T to remove unbound calponin and developing smooth muscle as well as in broblast cul-
the binding of calponin to F-actin was determined via tures is higher than that in adult smooth muscle tissues
the anti-h1 calponin mAb CP3 or the anti-h2 calponin [27,29]. This expression pattern is consistent with the
mAb CP21 at room temperature for 1h. Following nding that h2 calponin may play a role in regulating
washes as above, HRP-conjugated anti-mouse immu- cytokinesis [27] and be considered as a ‘‘cytoskeletal
noglobulin second antibody was incubated with the calponin.’’
236 J.-P. Jin et al. / Protein Expression and Purication 31 (2003) 231–239
Expression and purication of mouse h1 and h2 calponin after the cation and anion exchange columns at dif-
isoforms ferent pHs. A major contaminant protein with a mo-
lecular weight close to h1 calponin was then removed
DNA sequencing results veried that the h1 and h2 by high resolution Superose 12 FPLC gel ltration
calponin cDNAs cloned by RT-PCR from the 129SvJ chromatography. The purication of h2 calponin was
mouse, a strain often used in gene knockout studies, are relatively less complex and the anion exchange column
identical to those previously cloned from Balb/c mice [3]. was eective in terms of reducing contaminant proteins
Both h1 and h2 calponins were expressed at signicant to minimal levels. The following gel ltration using a
levels in E. coli culture (Fig. 3). The purication pro- conventional low pressure Sephadex G75 column was
cedures developed for the basic and neutral isoforms of able to purify the h2 calponin to homogeneity. The
calponin are very eective and highly puried mouse h1 dierent solubilities of h1 and h2 calponins in
and h2 calponin proteins were obtained (Fig. 3). The (NH4)2SO4 and their distinct behavior during the bio-
nal yields of the expression and purication of h1 and chemical fractionations reect their structural diver-
h2 calponins are 10 and 40 mg, respectively, per liter gence that may constitute a foundation for their
of bacterial culture. The results from amino acid anal- functional dierences.
ysis in Table 1 demonstrate a very good match between Addition of an anity tag such as the His-tag has
the residue ratios determined experimentally and that been widely used for facilitated purication of re-
predicted from amino acid sequences, reecting accurate combinant proteins [41]. However, there is a possibility
cloning and eective purication of the calponin iso- for the tag structure to modify the overall conformation
forms. Reproducibility of these methods has been con- of calponin that is a potentially allosteric regulatory
rmed. Since they are designed based on the physical protein and has been shown with conformational
property of the calponin isoforms, these protocols can changes sensitive to local structural modication [39].
be use for h1 and h2 calponins from other species, as we Therefore, we chose to express and purify h1 and h2
have recently succeeded in the expression and purica- calponins as non-fusion protein for a more reliable
tion of human h2 calponin (unpublished results). functional characterization.
The purication proles in Fig. 3 show that h1
calponin was separated from most bacterial proteins Structural diversity and similarities between h1 and h2
calponins
Using the puried mouse h1 and h2 calponin iso-
Table 1
Amino acid composition of cloned mouse h1 and h2 calponins form proteins, we have developed and characterized
several specic mAbs. Fig. 4 demonstrates the use-
Amino h1 Calponin h2 Calponin
acid fulness of these antibodies in identifying calponin
Ca Eb Ca Eb
isoforms by Western blot. The ELISA epitope titra-
Ala 18 17.7 21 22.0 tion curves shown in Fig. 5 demonstrate the structural
Arg 14 13.8 9 9.2 conservation as well as dierentiation of h1 and h2
Asx 33 33.0 32 32.6
calponins. mAbs CP1 and CP3 bind h1 calponin with
Gly 28 38.9c 34 38.8c
Glx 40 43.7 36 38.8 high anity and have no signicant cross-reaction to
His 11 11.1 5 6.6 h2 calponin (Fig. 5A). The cross-reaction of CP1 and
Ile 13 11.8 11 10.3 CP3 mAbs raised against chicken gizzard calponin
Leu 23 23.1 26 26.6 with mouse h1 calponin, but not h2 calponin, is
Lys 22 21.8 21 22.7
consistent with the phylogenetic relationship in which
Met 10 7.4 14 11.8
Phe 9 9.2 7 7.4 the chicken gizzard calponin belongs to the h1 cal-
Pro 14 15.4 14 18.0 ponin family (Fig. 1). mAb CP21, on the other hand,
Ser 15 11.3 24 22.2 has a high anity for h2 calponin and does not rec-
Thr 18 16.6 16 15.8 ognize h1 calponin (Fig. 5B). The presence of these
Tyr 12 11.7 13 12.8
isoform-specic epitopes indicates structural diver-
Val 12 12.5 12 11.7
Cys 3 NDd 8 NDd gence between the two calponin isoforms. The dier-
Trp 2 NDd 2 NDd ences in the tertiary structure of calponin isoforms
Total 297 299.2 305 307.5 suggest dierentiated functions. Nevertheless, the
a C, amino acid composition predicted from sequence data. RAH2 polyclonal antiserum raised against h2 calpo-
b E, amino acid composition experimentally determined from the nin binds h2 calponin with high avidity as expected
bacterially expressed protein.
and cross-reacts with h1 calponin with a lower avidity
c The unusually high Gly ratio may be due to a contamination of
(Fig. 5C). We have also developed several additional
the sample from gel electrophoresis reagents which were extensively
used in the laboratory and the protein facility. anti-h2 calponin mAbs that cross-react to h1 calponin
d ND, not determined. with various anities. In fact, only two of our 12
J.-P. Jin et al. / Protein Expression and Purication 31 (2003) 231–239 237
mAbs obtained by h2 calponin immunization have Similar binding anity of h1 and h2 calponins to F-actin
no cross-reaction to h1 calponin (data not shown).
The cross-reactions of polyclonal and monoclonal In addition to specic probes for epitope structure
antibodies to h1 and h2 calponins indicate epitopes analysis, the anti-h1 and anti-h2 calponin mAbs provide
shared by the two isoforms. Altogether, the results useful reagents for the ELISA protein binding analysis.
suggest that the epitopic structure of h1 and h2 cal- The results of solid phase protein binding assays showed
ponins is signicantly conserved, reecting conserved that h1 and h2 calponins bind F-actin with similar af-
three-dimensional structures. This hypothesis is in nity (Fig. 6). The actin-binding activity of mouse h1
agreement with the mainly conserved primary struc- and h2 calponins demonstrates the biological activity of
ture of the calponin isoforms (Fig. 1) and suggests the mammalian calponin protein expressed in bacterial
a conserved mechanism for their physiological func- cells. The binding anity detected for the mammalian
tion. calponins is similar to that previously demonstrated for
the chicken gizzard calponin [39]. The similarity in actin-
binding anity supports the hypothesis that h1 and h2
calponins may play a conserved function in regulating
the function of actin laments in dierent biological
processes. This hypothesis is supported further by the
primary structural alignment of h1 and h2 calponins
(Fig. 7), demonstrating largely conserved amino acid
sequences in the previously determined actin-binding
site of calponin [42]. Therefore, the rich information
obtained from previous studies on h1 calponin in
modulating smooth muscle contractility may be used to
guide investigations on the structure–function relation-
ship of h2 calponin for its function in regulating the
actin cytoskeleton in non-muscle cells.
The actin-binding anity of mouse h1 and h2 cal-
ponins expressed in E. coli is comparable with that pu-
ried from chicken gizzard tissue [39]. The exposure of
Fig. 6. Similar binding anity of h1 and h2 calponins for F-actin. calponin to 6M urea during purication did not aect
ELISA-mediated protein binding experiments were carried out to an-
the actin binding anity as compared with that of cal-
alyze the binding of mouse h1 and h2 calponins to immobilized
chicken gizzard F-actin. The titration curves show similar binding ponin proteins puried by a procedure using non-urea
anity of h1 and h2 calponins for F-actin. buers [33,39,43].
Fig. 7. Primary structure alignment of mouse h1 and h2 calponins. Amino acid sequences of mouse h1 and h2 calponins are aligned to demonstrate
their conserved and diverged sub-molecular structures. The diverged residues are shaded. The region previously determined for actin-binding is
outlined. The results show a higher degree of diversity in the COOH-terminal region, but the actin-binding region of the two calponin isoforms is
largely conserved.
238 J.-P. Jin et al. / Protein Expression and Purication 31 (2003) 231–239
Acknowledgments [17] W. Lehman, Calponin and the composition of smooth muscle thin
laments, J. Muscle Res. Cell Motil. 12 (1991) 221–224.
[18] A.P. Somlyo, A.V. Somlyo, Signal transduction by G-proteins,
We thank Mary Resek and Wenhua Chen for
rho-kinase and protein phosphatase to smooth muscle and non-
technical assistance, and Dr. M. Moazzem Hossain for muscle myosin II, J. Physiol. 522 (2000) 177–185.
the data shown in Fig. 4. This study was supported in [19] R. Nigam, C.R. Triggle, J.-P. Jin, h1- and h2-calponins are not
part by grants from the Medical Research Council of essential for norepinephrine- or sodium uoride-induced contrac-
Canada and March of Dimes Birth Defect Foundation tion of rat aortic smooth muscle, J. Muscle Res. Cell Motil. 19
(1998) 695–703.
to J-PJ.
[20] J.D. Matthew, A.S. Khromov, M.J. McDue, A.V. Somlyo, A.P.
Somlyo, S. Taniguchi, K. Takahashi, Contractile properties and
proteins of smooth muscles of a calponin knockout mouse,
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www.elsevier.com/locate/yprep
Expression and purication of the h1 and h2 isoforms of calponin
Jian-Ping Jin,a,b,* Di Wu,a,b Jimin Gao,b Rita Nigam,b and Stephen Kwongb
a Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4970, USA
b Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1
Received 25 March 2003, and in revised form 14 May 2003
Abstract
Three homologous calponin isoforms, named h1, h2, and acidic calponins, have been found in birds and mammals. Based
primarily on studies of chicken gizzard smooth muscle (h1) calponin, calponin has been identied as a family of actin-associated
proteins that inhibit actomyosin ATPase activity. Evolutionary divergence of the calponin isoforms suggests dierentiated function.
While the role of h1 calponin in smooth muscle contraction is under investigation, h2 calponin has been shown regulating the
function of actin cytoskeleton. Using cloned cDNA, we expressed mammalian h1 and h2 calponins in Escherichia coli. We have
developed eective methods to purify biologically active h1 and h2 calponin proteins from transformed bacterial culture. The
puried calponin isoform proteins were used to generate monoclonal antibodies that reveal epitopic structure dierence between h1
and h2 calponins. Together with their dierential expression in tissues and during development, the structural diversity of h1 and h2
calponins suggests non-redundant physiological function. Nevertheless, h1 and h2 calponins bind F-actin with similar anity,
indicating a conserved mechanism for their role in regulating actin laments in smooth muscle and non-muscle cells.
2003 Elsevier Science (USA). All rights reserved.
Calponin is a family of actin lament-associated lacking calponin is contractile upon physiological sim-
proteins found in smooth muscle and non-muscle cells. ulation, the expression of h1 calponin is correlated to
Encoded by homologous genes, three calponin isoforms, the sensitivity to norepinephrine activation [19]. The
h1 (basic) [1,2], h2 (neutral) [3], and acidic [4,5], have hypothesis that h1 calponin is a modulatory protein in
been identied. The physiological and pathological sig- smooth muscle contraction has been demonstrated fur-
nicance of the dierent calponin isoforms is largely ther in gene knockout mice, in which the lack of h1
unknown. h1 Calponin is the major calponin found in calponin in smooth muscle resulted in faster velocity of
smooth muscle and has been extensively analyzed in unloaded shortening [20,21]. While the physiological
many previous studies. In vitro protein binding studies function of h1 calponin in smooth muscle contraction
have demonstrated that calponin binds actin [6–8] and remains to be investigated, it has been shown that h1
tropomyosin [9,10], suggesting a role in the function of calponin is required for agonist-induced signal trans-
smooth muscle thin laments. h1 Calponin inhibits the duction in smooth muscle cells [22] and the survival of
actin-activated MgATPase activity of smooth muscle h1 calponin gene knockout mice may be based on
myosin [7,11–13], the movement of actin laments over compensation from changes in related proteins such as
immobilized myosin in in vitro motility assays [14,15], the level of actin [20].
and the force development and shortening velocity of The function of h2 calponin has not been extensively
skinned smooth muscle [16,17]. These data suggest that studied. Structural conservation suggests that the h1, h2,
in addition to the myosin-based regulation [18], h1 cal- and acidic calponins may function via similar molecular
ponin may play a modulatory role in smooth muscle mechanisms. On the other hand, the three calponin
contraction. Although rat vascular smooth muscle isoforms have signicantly diverged during vertebrate
evolution (Fig. 1). Although h1 and h2 calponins are
similar in size, their notable sequence diversity as dem-
* Corresponding author. Fax: 1-216-368-3952. onstrated by the dierent overall charges suggests that
E-mail address: jxj12@po.cwru.edu (J.-P. Jin). they have adapted to dierent biological processes. The
1046-5928/$ - see front matter 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S1046-5928(03)00185-2
232 J.-P. Jin et al. / Protein Expression and Purication 31 (2003) 231–239
indicating a conserved mechanism for their role in
regulating actin laments in smooth muscle and non-
muscle cells.
Materials and methods
Cloning of cDNAs encoding mouse h1 and h2 calponins by
reverse transcription-coupled polymerase chain reaction
We cloned a cDNA encoding mouse h1 calponin by
reverse transcription-coupled polymerase chain reaction
Fig. 1. Phylogenetic analysis of calponin isoforms. Amino acid se- (RT-PCR) from the uterus smooth muscle of pregnant
quences of human h1 (GenBank Accession No. S80560), h2 (D83735) 129SvJ mice. Total RNA was extracted from the fresh
and acidic (S80562) calponins, mouse h1 (Z19542) and h2 (Z19543) tissue with the TRIZOL reagent (Gibco-BRL) according
calponins, pig h1 (Z19538) and h2 (Z19539) calponins, rat h1 (X71071)
to manufacturers protocol. Using 2lg of the total RNA
and acidic (U06755) calponins, ferret h1 calponin (AF323674), and
chicken h1 calponin (M63559) were compared to construct a phylo- as template, an oligonucleotide primer complementary
genetic tree. The pI of each calponin isoform was calculated from to the sequence anking the translation stop codon of
amino acid sequences to demonstrate the dierences between the three mouse h1 calponin mRNA [3] was used for reverse
calponins. transcription of mouse h1 calponin cDNA. Double
stranded cDNA was then synthesized by PCR using this
expression of h1 calponin in smooth muscle is up-reg- primer paired with a primer corresponding to the se-
ulated during development, suggesting a function in quence anking the translation initiation codon of
mature myolaments [19,23–27]. In contrast, h2 calpo- mouse h1 calponin mRNA.
nin is expressed at higher levels in growing and remod- We cloned further a cDNA encoding mouse h2 cal-
eling smooth muscle tissues [27]. Expression of h2 ponin from RNA extracted from fresh stomach smooth
calponin was detected by RT-PCR in non-smooth muscle tissue of adult 129SvJ strain mouse. To amplify
muscle organs such as the heart [28]. We have recently the low abundant h2 calponin mRNA in stomach tissue,
shown high levels of h2 calponin expression in bro- a short oligo nucleotide primer complementary to a
blasts [29]. Over-expression of h1 [30] or h2 [27] calponin segment in the 30-untranslated region was used for the
inhibits the rate of cell proliferation, suggesting its role reverse transcription of rst strand cDNA. PCR was
in modulating actin-cytoskeleton during cytokinesis followed using a pair of primers corresponding to the
[27]. The potential role of calponin in non-contractile regions anking the translation initiation and termina-
function is supported by the observation that calponin tion codons, respectively, of the mouse h2 calponin
may participate in mitogen-activated protein kinase and mRNA [3].
protein kinase C signaling [31,32]. Restriction enzyme cloning sites were constructed
Comparative studies of h1 and h2 calponins can in the oligonucleotide primers. The double-stranded
provide useful information for understanding the phys- cDNA synthesized by PCR was digested with NdeI and
iological function of calponin. Biochemical studies using EcoRI and puried by the Prep-A-Gene glass bead-
puried protein form the foundation of functional binding method (Bio-Rad Laboratories) according to
characterization of calponin. Puried protein also pro- manufacturers protocol. The cDNA insert was ligated
vides standards for detecting calponin isoform expres- to NdeI–EcoRI-cut pAED4 prokaryotic expression
sion and materials for structural characterization. vector [34]. After transformation of JM109 strain of
Expression of cloned cDNA in bacteria is an eective Escherichia coli, ampicillin-resistant colonies containing
approach to prepare calponin isoform proteins. While the recombinant plasmids were identied by PCR for
the expression and purication of basic h1 calponin cDNA insert with appropriate sizes. The cloned h1 and
have been described in a procedure for chicken gizzard h2 calponin cDNAs were conrmed by DNA sequenc-
calponin [33], the method for expression and purica- ing using dideoxy chain termination method as de-
tion of h2 calponin has not been reported. In the present scribed previously [35].
study, we developed eective methods for the expression
and purication of mammalian h1 and h2 calponins Expression and purication of mouse h1 and h2 calponins
from cloned cDNA. The puried calponin isoforms
were used to develop specic monoclonal antibodies The procedures were carried out at 4 °C unless spec-
(mAbs) that reveal dierent epitopic structures in h1 ied.
and h2 calponins. Protein binding assays showed that The recombinant pAED4 expression plasmids were
h1 and h2 calponins bind F-actin with similar anity, used to transform BL21(DE3)pLysS strain of E. coli [36]
J.-P. Jin et al. / Protein Expression and Purication 31 (2003) 231–239 233
for the expression of mouse h1 or h2 calponin proteins. linear KCl gradient and the h1 calponin fractions were
Two times tryptone–yeast broth containing 100 lg/mL identied by SDS–PAGE, dialyzed, and concentrated by
ampicillin and 25 lg/mL chloramphenicol was inocu- lyophilization. The protein was then dissolved in a small
lated with a single colony of the freshly transformed volume of 6M urea, 0.2 M KCl, and 10mM imidazole–
bacteria and incubated at 37 °C with vigorous shaking. HCl, pH 7.0, and fractionated on a preparative Supe-
When the OD600 reached 0.4–0.5, the cultures were in- rose 12 gel ltration column for FPLC to remove the
duced with 0.4 mM isopropyl-1-thio-b-D-galactopyr- remaining contamination protein with a molecular
anoside (IPTG) and incubated further for 3h. The weight close to that of h1 calponin (Fig. 3). The column
bacteria were harvested by centrifugation, re-suspended fractions were examined by SDS–PAGE and those
in 50 mL of 50 mM Tris–HCl buer, pH 8.0, 2.5 mM containing pure h1 calponin were lyophilized after di-
EDTA, and lysed by three passes through a French alysis against 100 volumes of 0.5% formic acid for three
press at 1000 psi. The lysate was centrifuged to remove changes.
the insoluble materials and fractionated by step am- The h2 calponin in the bacterial lysate was enriched
monium sulfate precipitation. Predicted from the amino in the ammonium sulfate fraction of 0–30% saturation
acid sequences, the h1 and h2 isoforms of calponin have
signicantly dierent biochemical properties (Fig. 2).
Therefore, dierent procedures have been developed for
their purication.
As shown in Fig. 3, h1 calponin was present in the
ammonium sulfate fraction of 20–40% saturation. After
dialysis to remove the salt, urea was added to 6M and
the pH was adjusted to 7.0 in 20 mM imidazole–HCl
buer containing 0.1 mM EDTA and 6mM b-mercap-
toethanol for ion-exchange chromatography fraction-
ation of the basic h1 calponin (isoelectric point (pI) 9)
on a CM52 cation-exchange column. The protein solu-
tion was centrifuged to remove a small amount of pre-
cipitates and loaded onto the column equilibrated in 6M
urea, 0.1 mM EDTA, 6mM b-mercaptoethanol, and
10 mM imidazole–HCl, pH 7.0. After washing, the col-
umn-bound proteins were eluted with a 0–500 mM linear
Fig. 3. Bacterial expression and purication of mouse h1 and h2 cal-
KCl gradient in the same buer and the fractions were ponins. The recombinant expression plasmids encoding mouse h1 and
analyzed by SDS–polyacrylamide gel electrophoresis h2 calponins were used to transform BL21(DE3)pLysS E. coli. The
(SDS–PAGE). The protein peak containing h1 calponin transformed bacteria were cultured in liquid media at large scales and
was dialyzed to remove salt and re-fractionated on a induced with IPTG. The h1 and h2 calponins expressed in E. coli were
puried by serial fractionations using ammonium sulfate precipitation,
DE52 anion exchange column in 6M urea, 0.1 mM
ion-exchange, and gel ltration chromatographies. The SDS–PAGE
EDTA, 6mM b-mercaptoethanol, and 20 mM Tris– gels summarize the results of expression, each step of purication, and
HCl, pH 10. The column was eluted with a 0–300 mM nal products of the calponin isoform proteins.
Fig. 2. Predicted charge prole of h1 and h2 calponins. The calculated charge proles of mouse h1 and h2 calponins against dierent pH are shown.
The predicted charge property was used to direct designing ion-exchange chromatography purication of the basic and neutral calponin isoforms.
234 J.-P. Jin et al. / Protein Expression and Purication 31 (2003) 231–239
(Fig. 3). After dialysis to remove the salt, urea was were examined by SDS–PAGE and those containing
added to 6 M and the pH was adjusted to 8.8 in 10 mM pure h2 calponin (Fig. 3) were lyophilized after dialysis
Tris–HCl buer, 0.1 mM EDTA, and 6mM b-mercap- against 0.5% formic acid as above.
toethanol. After clarication by centrifugation, the The puried mouse h1 and h2 calponins were veried
protein solution containing the neutral h2 calponin by Western blotting using specic antibodies as previ-
(pI 7.6) was loaded on a DE52 anion exchange column ously described [19] and by amino acid analysis at the
for chromatography fractionation. After washing away Protein Sequencing Facility, University of Calgary.
the unbound proteins, the column was eluted with a 0–
300 mM linear KCl gradient in the same buer. The h2 Anti-calponin antibodies
calponin peak was identied by SDS–PAGE, dialyzed to
remove urea and salt, and lyophilized as above. To Two mouse mAbs (CP1 and CP3, IgG1j and Ig-
purify further the h2 calponin to homogeneity, the G2bj, respectively) previously developed by immuni-
preparation was fractionated further on a Sephadex G75 zation with puried chicken gizzard calponin [37] were
gel ltration column equilibrated in 6M urea, 0.2 M used in the present study. Both CP1 and CP3 cross-react
KCl, 6 mM b-mercaptoethanol, and 10 mM imidazole with mammalian h1 calponin but not h2 calponin (Figs.
buer, pH 7.0. Fractions from the gel ltration column 4 and 5).
The puried mouse h2 calponin protein was used as
immunogen for the preparation of mAbs against h2
calponin. The immunization of Balb/c mice and hy-
bridoma fusion were performed as described previously
[27,37]. The hybridomas were screened by enzyme-
linked immunosorbent assay (ELISA) against puried
h2 calponin using anti-mouse total immunoglobulin
second antibody for clones secreting anti-h2 calponin
mAb. Despite the weak immunogenicity of mouse h2
calponin in immunizing mice, we were able to obtain
signicant anti-h2 calponin antibody titer in the mouse
sera. Multiple hybridoma cell lines secreting high af-
nity anti-h2 calponin mAb were obtained, although
most of them produced IgM. The mAb specicity was
examined by Western blotting against puried h1 and
h2 calponins. Using Western blots transferred from
SDS–PAGE that resolves the size dierence between h1
and h2 calponins, the mAb specicity was veried on
Fig. 4. Dierential mobility of h1 and h2 calponins in SDS–PAGE and adult mouse urinary bladder tissue expressing both
specicity of anti-calponin isoform antibodies. The SDS–PAGE shows calponin isoforms [27]. An h2 calponin-specic mAb,
dierential mobility of mouse h1 (partially puried) and h2 calponins
CP21 (IgMj) [27], was used in the present study.
together with total protein homogenates of 3-day-old mouse urinary
bladder sample. The Western blots demonstrate the specicity of the The puried mouse h2 calponin was also used to
four anti-calponin antibodies used in this study. immunize a New Zealand White rabbit to produce
Fig. 5. Epitope analysis of h1 and h2 calponin isoforms. ELISA titration was carried out to compare the diversity as well as conservation between the
epitopic structure of h1 and h2 calponins. The gures show the titration curves for the anti-h1 mAbs CP1 and CP3 (A), the anti-h2 calponin mAb
CP21 (B), and the RAH2 rabbit antiserum (C). The results show the presence of h1 and h2 calponin-specic epitopes recognized by the mAbs.
Nevertheless, the titration curves of RAH2 polyclonal antibody against h1 and h2 calponins demonstrate the presence of shared epitopes in the two
calponin isoforms.
J.-P. Jin et al. / Protein Expression and Purication 31 (2003) 231–239 235
polyclonal antisera. The rabbit was injected intramus- plate at room temperature for 45 min followed by nal
cularly with 1 mg of the h2 calponin immunogen mixed washes and H2O2–ABTS substrate colour reaction.
in Freunds complete adjuvant and boosted three times The A405 nm of triplicate assay wells was monitored at a
with the same amounts of antigen in Freunds incom- series of time points using automated microplate
plete adjuvant at three-week intervals. Upon the detec- reader. The titration curves were plotted against the
tion of high serum antibody titer against h2 calponin, concentrations of calponin and the statistical signi-
the rabbit was sacriced to collect the immune serum cance of the dierences was examined by Students
(RAH2) [19]. t test.
ELISA epitope analysis Protein sequence analysis
We applied antibody epitope anity titration to ex- The charge proles of h1 and h2 calponins were
amine the structural diversity and conservation of h1 predicted using the Protean computer program
and h2 calponins. The epitope analysis was carried out (DNAStar). Phylogenetic analysis of calponin isoforms
by ELISA as described previously [38,39]. The puried and primary structure alignment for mouse h1 and h2
mouse h1 and h2 calponins were dissolved in Buer A calponins were done by the Clustal method using the
(0.1 M KCl, 10 mM piperazine-N,N0-bis(2-ethanesulf- MegAlign program (DNAStar).
onic acid) (PIPES), pH 7.0, and 3 mM MgCl2), at equal
concentrations for coating on microtiter plates. After
washing and blocking using phosphate-buered saline Results and discussion
(PBS) containing 0.05% Tween 20 (PBS-T), the plates
were incubated with serial dilutions of anti-calponin Evolutionary divergence of calponin isoforms
antibodies in PBS-T containing 0.1% bovine serum al-
bumin (BSA), followed by reactions with horseradish The phylogenetic tree in Fig. 1 demonstrates the
peroxidase (HRP)-conjugated anti-mouse total immu- evolutionary relationships among h1, h2, and acidic
noglobulin or anti-rabbit IgG second antibody and calponins in avian and mammalian species. The results
H2O2/2,20-azinobis-(3-ethylbenzthiazolinesulfonic acid show that the primary structure of each calponin iso-
(ABTS) substrate. The binding between the anti-calpo- form in birds and mammals is more conserved than
nin antibody and the calponin isoforms was quantied those of the three calponin isoforms in each species. The
by A405nm readings recorded at a series of time points by relationships indicate that the three calponin isoforms
an automated microplate reader (Benchmark, Bio-Rad). diverged early during vertebrate evolution. This phylo-
The A405nm readings from a time point within the linear genetic pattern is similar to that among the three tro-
range of the color development were used to construct ponin T (TnT) genes encoding cardiac, slow, and fast
the titration curves. The titrations were done in tripli- skeletal muscle TnT [40], suggesting a conserved func-
cate. tion of the calponin isoforms in dierent cell types. It is
interesting to note that the structure of h2 calponin
ELISA-based solid-phase actin-binding experiments (neutral) has diverged signicantly from that of h1 cal-
ponin (basic) and the acidic calponin may present an
ELISA-based protein-binding experiments [39] were intermediate isoform (Fig. 1). In other words, the
carried out to characterize the interactions of h1 and smooth muscle-specic h1 calponin is evolutionarily
h2 calponins with F-actin. Microtiter plates were more distant from h2 calponin than from acidic calpo-
coated at 4 °C overnight with smooth muscle F-actin nin. The structural dierence between h1 and h2 cal-
puried from chicken gizzard as described previously ponins may suggest specic adaptations to their activity
[39] (30 lg/ml and 0.1 ml/well) in Buer A as described and cellular environment. H1 calponin is the major
above. After three washes to remove the excess actin calponin found in smooth muscle [19,37] and has been
and block the remaining plastic surface with Buer A investigated as a potential modulator of smooth muscle
containing 0.05% Tween 20 (Buer T), the plates were contraction in many previous studies. In contrast, h2
incubated with serial dilutions of mouse h1 or h2 calponin is much less abundant in smooth muscle and
calponin in Buer T containing 0.1% BSA at room also expressed in certain non-muscle cells such as -
temperature for 2h. The plates were then washed three broblasts [29]. The level of h2 calponin expressed in
times with Buer T to remove unbound calponin and developing smooth muscle as well as in broblast cul-
the binding of calponin to F-actin was determined via tures is higher than that in adult smooth muscle tissues
the anti-h1 calponin mAb CP3 or the anti-h2 calponin [27,29]. This expression pattern is consistent with the
mAb CP21 at room temperature for 1h. Following nding that h2 calponin may play a role in regulating
washes as above, HRP-conjugated anti-mouse immu- cytokinesis [27] and be considered as a ‘‘cytoskeletal
noglobulin second antibody was incubated with the calponin.’’
236 J.-P. Jin et al. / Protein Expression and Purication 31 (2003) 231–239
Expression and purication of mouse h1 and h2 calponin after the cation and anion exchange columns at dif-
isoforms ferent pHs. A major contaminant protein with a mo-
lecular weight close to h1 calponin was then removed
DNA sequencing results veried that the h1 and h2 by high resolution Superose 12 FPLC gel ltration
calponin cDNAs cloned by RT-PCR from the 129SvJ chromatography. The purication of h2 calponin was
mouse, a strain often used in gene knockout studies, are relatively less complex and the anion exchange column
identical to those previously cloned from Balb/c mice [3]. was eective in terms of reducing contaminant proteins
Both h1 and h2 calponins were expressed at signicant to minimal levels. The following gel ltration using a
levels in E. coli culture (Fig. 3). The purication pro- conventional low pressure Sephadex G75 column was
cedures developed for the basic and neutral isoforms of able to purify the h2 calponin to homogeneity. The
calponin are very eective and highly puried mouse h1 dierent solubilities of h1 and h2 calponins in
and h2 calponin proteins were obtained (Fig. 3). The (NH4)2SO4 and their distinct behavior during the bio-
nal yields of the expression and purication of h1 and chemical fractionations reect their structural diver-
h2 calponins are 10 and 40 mg, respectively, per liter gence that may constitute a foundation for their
of bacterial culture. The results from amino acid anal- functional dierences.
ysis in Table 1 demonstrate a very good match between Addition of an anity tag such as the His-tag has
the residue ratios determined experimentally and that been widely used for facilitated purication of re-
predicted from amino acid sequences, reecting accurate combinant proteins [41]. However, there is a possibility
cloning and eective purication of the calponin iso- for the tag structure to modify the overall conformation
forms. Reproducibility of these methods has been con- of calponin that is a potentially allosteric regulatory
rmed. Since they are designed based on the physical protein and has been shown with conformational
property of the calponin isoforms, these protocols can changes sensitive to local structural modication [39].
be use for h1 and h2 calponins from other species, as we Therefore, we chose to express and purify h1 and h2
have recently succeeded in the expression and purica- calponins as non-fusion protein for a more reliable
tion of human h2 calponin (unpublished results). functional characterization.
The purication proles in Fig. 3 show that h1
calponin was separated from most bacterial proteins Structural diversity and similarities between h1 and h2
calponins
Using the puried mouse h1 and h2 calponin iso-
Table 1
Amino acid composition of cloned mouse h1 and h2 calponins form proteins, we have developed and characterized
several specic mAbs. Fig. 4 demonstrates the use-
Amino h1 Calponin h2 Calponin
acid fulness of these antibodies in identifying calponin
Ca Eb Ca Eb
isoforms by Western blot. The ELISA epitope titra-
Ala 18 17.7 21 22.0 tion curves shown in Fig. 5 demonstrate the structural
Arg 14 13.8 9 9.2 conservation as well as dierentiation of h1 and h2
Asx 33 33.0 32 32.6
calponins. mAbs CP1 and CP3 bind h1 calponin with
Gly 28 38.9c 34 38.8c
Glx 40 43.7 36 38.8 high anity and have no signicant cross-reaction to
His 11 11.1 5 6.6 h2 calponin (Fig. 5A). The cross-reaction of CP1 and
Ile 13 11.8 11 10.3 CP3 mAbs raised against chicken gizzard calponin
Leu 23 23.1 26 26.6 with mouse h1 calponin, but not h2 calponin, is
Lys 22 21.8 21 22.7
consistent with the phylogenetic relationship in which
Met 10 7.4 14 11.8
Phe 9 9.2 7 7.4 the chicken gizzard calponin belongs to the h1 cal-
Pro 14 15.4 14 18.0 ponin family (Fig. 1). mAb CP21, on the other hand,
Ser 15 11.3 24 22.2 has a high anity for h2 calponin and does not rec-
Thr 18 16.6 16 15.8 ognize h1 calponin (Fig. 5B). The presence of these
Tyr 12 11.7 13 12.8
isoform-specic epitopes indicates structural diver-
Val 12 12.5 12 11.7
Cys 3 NDd 8 NDd gence between the two calponin isoforms. The dier-
Trp 2 NDd 2 NDd ences in the tertiary structure of calponin isoforms
Total 297 299.2 305 307.5 suggest dierentiated functions. Nevertheless, the
a C, amino acid composition predicted from sequence data. RAH2 polyclonal antiserum raised against h2 calpo-
b E, amino acid composition experimentally determined from the nin binds h2 calponin with high avidity as expected
bacterially expressed protein.
and cross-reacts with h1 calponin with a lower avidity
c The unusually high Gly ratio may be due to a contamination of
(Fig. 5C). We have also developed several additional
the sample from gel electrophoresis reagents which were extensively
used in the laboratory and the protein facility. anti-h2 calponin mAbs that cross-react to h1 calponin
d ND, not determined. with various anities. In fact, only two of our 12
J.-P. Jin et al. / Protein Expression and Purication 31 (2003) 231–239 237
mAbs obtained by h2 calponin immunization have Similar binding anity of h1 and h2 calponins to F-actin
no cross-reaction to h1 calponin (data not shown).
The cross-reactions of polyclonal and monoclonal In addition to specic probes for epitope structure
antibodies to h1 and h2 calponins indicate epitopes analysis, the anti-h1 and anti-h2 calponin mAbs provide
shared by the two isoforms. Altogether, the results useful reagents for the ELISA protein binding analysis.
suggest that the epitopic structure of h1 and h2 cal- The results of solid phase protein binding assays showed
ponins is signicantly conserved, reecting conserved that h1 and h2 calponins bind F-actin with similar af-
three-dimensional structures. This hypothesis is in nity (Fig. 6). The actin-binding activity of mouse h1
agreement with the mainly conserved primary struc- and h2 calponins demonstrates the biological activity of
ture of the calponin isoforms (Fig. 1) and suggests the mammalian calponin protein expressed in bacterial
a conserved mechanism for their physiological func- cells. The binding anity detected for the mammalian
tion. calponins is similar to that previously demonstrated for
the chicken gizzard calponin [39]. The similarity in actin-
binding anity supports the hypothesis that h1 and h2
calponins may play a conserved function in regulating
the function of actin laments in dierent biological
processes. This hypothesis is supported further by the
primary structural alignment of h1 and h2 calponins
(Fig. 7), demonstrating largely conserved amino acid
sequences in the previously determined actin-binding
site of calponin [42]. Therefore, the rich information
obtained from previous studies on h1 calponin in
modulating smooth muscle contractility may be used to
guide investigations on the structure–function relation-
ship of h2 calponin for its function in regulating the
actin cytoskeleton in non-muscle cells.
The actin-binding anity of mouse h1 and h2 cal-
ponins expressed in E. coli is comparable with that pu-
ried from chicken gizzard tissue [39]. The exposure of
Fig. 6. Similar binding anity of h1 and h2 calponins for F-actin. calponin to 6M urea during purication did not aect
ELISA-mediated protein binding experiments were carried out to an-
the actin binding anity as compared with that of cal-
alyze the binding of mouse h1 and h2 calponins to immobilized
chicken gizzard F-actin. The titration curves show similar binding ponin proteins puried by a procedure using non-urea
anity of h1 and h2 calponins for F-actin. buers [33,39,43].
Fig. 7. Primary structure alignment of mouse h1 and h2 calponins. Amino acid sequences of mouse h1 and h2 calponins are aligned to demonstrate
their conserved and diverged sub-molecular structures. The diverged residues are shaded. The region previously determined for actin-binding is
outlined. The results show a higher degree of diversity in the COOH-terminal region, but the actin-binding region of the two calponin isoforms is
largely conserved.
238 J.-P. Jin et al. / Protein Expression and Purication 31 (2003) 231–239
Acknowledgments [17] W. Lehman, Calponin and the composition of smooth muscle thin
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[18] A.P. Somlyo, A.V. Somlyo, Signal transduction by G-proteins,
We thank Mary Resek and Wenhua Chen for
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technical assistance, and Dr. M. Moazzem Hossain for muscle myosin II, J. Physiol. 522 (2000) 177–185.
the data shown in Fig. 4. This study was supported in [19] R. Nigam, C.R. Triggle, J.-P. Jin, h1- and h2-calponins are not
part by grants from the Medical Research Council of essential for norepinephrine- or sodium uoride-induced contrac-
Canada and March of Dimes Birth Defect Foundation tion of rat aortic smooth muscle, J. Muscle Res. Cell Motil. 19
(1998) 695–703.
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