Protein Expression and PuriWcation 42 (2005) 219–225

www.elsevier.com/locate/yprep

Expression, puriWcation, and functional testing

of recombinant CYR61/CCN1
Norbert Schütze a,¤, Karin Kunzi-Rapp b, Rita Wagemanns a, Ulrich Nöth a,
Susanne Jatzke a, Franz Jakob a
a
Orthopaedic Department, Molecular Orthopaedics, University of Würzburg, Germany
b
Institute for Laser Technologies and Metrology, University of Ulm, Germany

Received 1 February 2005, and in revised form 29 March 2005

Available online 19 April 2005

Abstract

The human cysteine-rich protein 61 (CYR61/CCN1) belongs to the CCN family of genes which plays an important role in cellular
processes such as proliferation, migration, adhesion, and diVerentiation. These extracellular matrix signaling molecules consist of a
modular structure and contain 38 conserved cysteine residues. Previously, we have shown that CYR61 is expressed in human osteo-
blasts and is regulated by bone-relevant growth factors. The protein also plays a role in angiogenesis. The open reading frame was
cloned into a baculovirus expression vector and transfected into SF-21 insect cells. Recombinant protein was expressed as a fusion
protein with the Fc-domain of human IgG and puriWed using aYnity chromatography on protein G–Sepharose columns. The cho-
rioallantoic membrane assay veriWed that blood vessel formation was stimulated by rCYR61. Additionally, human primary mesen-
chymal stem cells, osteoblasts, and endothelial cells responded to CYR61 treatment by a markedly stimulated proliferation.
rCYR61-Fc represents a tool to elucidate its role in cells of the bone microenvironment.
 2005 Elsevier Inc. All rights reserved.

Keywords: CCN family; CYR61(CCN1); Recombinant CYR61-Fc protein; Angiogenesis; Osteoblasts; Mesenchymal stem cells

Members of the CCN family of the cysteine-rich pro-
tein 61 (CYR61/CCN1),1 connective tissue growth factor
(CTGF/CCN2), and the nephroblastoma overexpressed
protein (nov/CCN3) function in processes such as prolif-
eration, migration, adhesion, and diVerentiation primar-
ily in bone, muscle, vasculature, and the nervous system
[1–3]. The proteins represent secreted matrix associated
signal molecules and share a common modular structure
including 38 strictly conserved cysteine residues (except
for CCN5 lacking the C-terminal module). The family
*
Corresponding author. Fax: +49 931 803 3599.
E-mail address: n-schuetze.klh@mail.uni-wuerzburg.de (N. Schütze).
1 actions, either oligomerization (VWC) or interactions
Abbreviations used: CYR61, cysteine-rich protein 61; CTGF, con-
nective tissue growth factor; IGFBP, insulin-like growth factor-bind-
ing protein; VWC, von Willebrand type C domain; VEGFc, vascular
endothelial growth factor c.
consists of six structurally related members which pos-
sess 30–50% homology at the amino acid level [4,5]. Each
domain is encoded by a separate exon [6]. N-terminal
amino acids have been shown to be important for secre-
tion of CYR61 [7]. Although CCN proteins share an
insulin-like growth factor-binding protein (IGFBP)-like
motif close to the N terminus, no clear experimental sig-
niWcance exists to suggest a function in the IGF signal-
ing pathway [8]. The von Willebrand type C domain
(VWC), the thrombospondin type I domain, and the
C-terminal module (which is absent in WISP2/CCN5)
are considered to be important for protein–protein inter-
with extracellular matrix molecules and receptors. Inter-
action partners of CCN proteins include integrin
receptors [9–15]. Additionally, less characterized motifs

1046-5928/$ - see front matter  2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.pep.2005.03.031
220 N. Schütze et al. / Protein Expression and PuriWcation 42 (2005) 219–225
could play a role for interactions with other binding
partners partly due to the heparin-binding of CYR61.
An important target for CCN proteins is bone and car-
tilage due to the respective expression pattern of CCN
members in animal models and human tissues, and the
majority of the above-mentioned receptors and pathways
are also relevant to skeletal homeostasis. CYR61 is
involved in chondrogenesis in mice [16], is expressed in
human bone at sites of bone remodeling, in hypertrophic
chondrocytes at the growth plate [17], and in the fracture
callus in rats [18]. In human osteoblasts, CYR61 expression
is regulated by a variety of bone-relevant growth factors
[19]. At the molecular level, recombinant CYR61 protein
was used to elucidate a genetic program for wound healing
in Wbroblasts [5]. Additionally, CYR61 is important for
angiogenesis, since a mouse model with functional inacti-
vation of the CYR61 gene is characterized by angiogenesis
problems such as a disrupture of the vascular integrity and
an impaired expression of vascular endothelial growth fac-
tor c (VEGFc) [20]. Also angiogenesis assays point towards
proangiogenic actions of CYR61 [21]. A general interplay
between angiogenesis and bone repair was recently
reviewed by Carano and FilvaroV [22]. In this respect it is
interesting to note that in addition to the above-mentioned
actions of CYR61 in angiogenesis and bone, FGF-2 (an
angiogenic growth factor) largely stimulates CYR61 at the
mRNA and the protein level [19]. CYR61 could play an
integrative role in these processes. An important tool to
elucidate the function of CYR61 at the molecular level is
the recombinant production of functional protein. Here,
we describe the expression, puriWcation, and functional
testing of CYR61 protein from insect cells.
Materials and methods
Materials
FCS was supplied by Gibco (Eggenstein, Germany).
SF-21 insect cells and a baculovirus expression system
were purchased from Clontech (Heidelberg, Germany).
All other chemicals were of the highest purity available.
Cell culture
SF-21 insect cells were cultured in medium (Invitro-
gen) at 25 °C. Osteoblasts (hFOB-cell line) [23] and
endothelial cells (EA hy 926) [24] were maintained in
DMEM/F12 medium containing 10% FCS at 37 °C in
5% CO2. Medium was changed twice per week.
Cloning for expression analysis
The open reading frame for CYR61 was ampliWed
from total RNA prepared from hFOB cells treated with
TNF
for 1 h, since previous studies have shown a high
induction of CYR61 mRNA at this condition [19]. Prim-
ers contained restriction sites at the 5⬘ end. The PCR
product was subcloned into a TA-TOPO vector (pCR
2.1, Invitrogen). The insert was prepared by double
restriction (EcoRI/BamHI) and cloned into the transfer
vector pBakPak 8 (Clontech, Heidelberg, Germany). The
Fc-domain of human IgG was ampliWed from a PC3
expression vector (kindly provided by Dr. Pascal Schnei-
der, University of Lausanne, Switzerland) by PCR and
subcloned 3⬘ to the open reading frame of CYR61 to
generate the recombinant vector.
Transfection of insect cells and expression of rCYR61
SF-21 caterpillar (lepidopteran) derived cells were
used to express recombinant CYR61 protein (rCYR61).
The recombinant vector was mixed with linearized DNA
which contains a modiWed Autographa californica
multiple nucleopolyhedrovirus (AcMNPV) genome. SF-
21 cells were transfected according to the suppliers infor-
mation in wells of six-well plates. After 5 days at 25 °C in
serum containing medium the supernatant (passage 1)
was used to infect SF-21 cells in 25 cm2 Xasks using
serum-free culture conditions. After 4 days, the superna-
tants (passage 2) were checked for protein expression and
further ampliWed one round using 75 cm2 Xasks to gener-
ate the Wnal stock (passage 3), which was stored in
aliquots at ¡20 °C for further puriWcations of the
protein.
PuriWcation of the rCYR61 protein
To purify the rCYR61 protein, protein G–Sepharose
columns (1 ml columns) were used with an Atka-FPLC
system (Amersham Pharmacia, Freiburg, Germany).
Columns were equilibrated with pH 7 buVered PBS, the
supernatant was applied (volumes between 15 and 45 ml)
at a Xow rate of 2 ml/min. Columns were washed with 10
column volumes of PBS and the protein was eluted with
elution buVer (0.1 M glycine, pH 2.5). Directly thereafter
eluted fractions were neutralized using 3 M Tris/HCl, pH
8. The rCYR61 protein was desalted for the angiogenesis
assay using a PD-10 column (Amersham Pharmacia,
Freiburg, Germany) and the yield was determined by a
conventional Bradford protein assay. The purity of the
protein Wnally was checked by silver gel electrophoresis
and Western blotting.
Silver gel electrophoresis and Western blotting
Silver gel electrophoresis and Western blotting essen-
tially were performed as described previously [19]. A
polyclonal antiserum against the full-length human
CYR61-Fc fusion protein was raised in rabbits and sub-
sequently the IgG fraction was aYnity-puriWed via pro-
tein G–Sepharose columns.
 N. Schütze et al. / Protein Expression and PuriWcation 42 (2005) 219–225
Angiogenesis assay
The chorioallantoic membrane (CAM) assay was per-
formed according to Kunzi-Rapp et al. [25]. BrieXy, fer-
tilized chicken eggs were purchased from Zeh, K.G.,
Laichingen, Germany, and stored at 60% humidity at
37 °C. The eggs were opened on the apical pole of the egg
shell on day 5 of hatching. Part of the membrane was
exposed by cutting a circular window with 3 cm in diam-
eter. The window was covered with a tape and incuba-
tion was continued. On day 7 of hatching, two silicone
rings with a thickness of 0.5 mm and an inner diameter
of 6 mm were placed on each membrane. On day 8,
rCYR61 solution (50 ng–1 g in a volume of 10 l in
buVer PBS) was applied into one of the silicone rings
placed on the CAM surface. The second ring with buVer
alone served as a control. Vascular growth in the ring
Welds and viability of the embryo were documented daily
until day 11 by video microscopy at diVerent magniWca-
tions (18£ and 25£) using a Zeiss Universal S3B operat-
ing microscope (Zeiss, Oberkochen, Germany) and
images were recorded by a Sony MC-3249 CCD camera
using Visupac version 22.1 software (Zeiss, Oberkochen,
Germany). Six CAMs were studied for each test group
and the experiments were repeated at least three times.
Isolation of mesenchymal stem cells
Mesenchymal stem cells (MSCs) were isolated from
human bone marrow obtained from the femoral head of
patients undergoing total hip arthroplasty using a modi-
Wed protocol [26] originally described by Haynesworth
et al. [27]. Usage of patient material was approved by the
Local Ethics Committee of the University of Würzburg
and written consent was obtained from each patient. Tra-
becular bone pieces were harvested from the cutting plane
of the femoral head and transferred to 50 ml conical tubes
containing DMEM/F12 medium (PAA, Cölbe, Ger-
many). After vortexing and centrifugation (1000 rpm,
5 min), the pellet containing bone pieces and released cells
was reconstituted in DMEM/F12 medium supplemented
with 10% fetal bovine serum (FBS), antibiotics (50 IU
penicillin/ml and 50 g streptomycin/ml), and 50 g/ml
ascorbate (complete medium). After repeated washings,
the released cells were pelleted (1000 rpm, 5 min), suspended
in complete medium, plated at a density of 60 £106 cells per
150 cm2 tissue culture Xask, and maintained at 37 °C in 5%
CO2. Non-adherent cells were removed after 2 days and
subsequently the medium was changed every 2 days until
the cell cultures reached conXuency.
Proliferation analysis
The WST-1 test was used to measure proliferation of
MSCs, osteoblasts (hFOB-cell line, kindly provided by
Prof. T.C. Spelsberg, Roster, USA) [23], and endothelial
221
cells (EA hy 926-cell line) [24]. BrieXy, 4000 cells were
seeded per well of a 96-well microplate. After 1 day, the
medium was changed to serum-free medium. After 24 h,
cells received rCYR61 (500 ng/ml) or buVer. Twelve
wells were treated similarly. After 24 h the substrate was
added for 4 h. Thereafter the absorbance was measured
in an ELISA reader at 450 nm.
Results
PuriWcation of CYR61
The recombinant CYR61 protein was expressed as an
IgG-Fc-tagged protein (rCYR61) from baculovirus-
infected SF-21 insect cells. Passage 2 supernatants were
positive in Western-blot analysis (data not shown) and
were subsequently used to infect SF-21 cells for FPLC
puriWcation. The elution proWle resulted in a single peak
(Fig. 1, Table 1). From two 150 cm2 Xasks, the FPLC
Fig. 1. FPLC-puriWcation proWle. Supernatants from baculovirus-
infected SF-21 insect cells (30 ml) were applied to a protein G–Sepharose
column at a Xow rate of 2 ml/min using an Atka-FPLC system. Using
0.1 M glycine (pH 2.5) the rCYR61 protein was eluted and subsequently
neutralized and analyzed for yield and purity.
Table 1
Amino acid sequence of the rCYR61-Fc fusion protein
MSSRIARALALVVTLLHLTRLALSTCPAACHCPLEAPKCAPG
VGLVRDGCGCCKVCAKQLNEDCSKTQPCDHTKGLECNFGA
SSTALKGICRAQSEGRPCEYNSRIYQNGESFQPNCKHQCTCI
DGAVGCIPLCPQELSLPNLGCPNPRLVKVTGQCCEEWVCDE
DSIKDPMEDQDGLLGKELGFDASEVELTRNNELIAVGKGSS
LKRLPVFGMEPRILYNPLQGQKCIVQTTSWSQCSKTCGTGIS
TRVTNDNPECRLVKETRICEVRPCGQPVYSSLKKGKKCSKT
KKSPEPVRFTYAGCLSVKKYRPKYCGSCVDGRCCTPQLTRT
VKMRFRCEDGETFSKNVMMIQSCKCNYNCPHANEAAFPFY
RLFNDIHKFRDEFGSVDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQVNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Amino acids of the rCYR61-Fc protein are shown in the one letter
code. The Fc-tag is connected C-terminal to the open reading frame of
the human CYR61 protein via a linker sequence shown in bold.
222 N. Schütze et al. / Protein Expression and PuriWcation 42 (2005) 219–225

brane (CAM) assay was applied to prove the functional-

ity of rCYR61. PuriWed rCYR61 was added to egg
membranes within a small silicone ring at 50 ng and 1 g
amounts which resulted in a clear induction of blood
vessel formation after 4 days (Fig. 3).

Proliferation

CCN proteins are considered to induce proliferation.

Fig. 2. Purity of CYR61. To check the purity of rCYR61 silver gel elec-
trophoresis and Western blotting were applied using standard proce-
dures as described in Materials and methods. (A) Western-blot analysis
and (B) silver gel electrophoresis. Size of the fusion protein is 73 kDa.
puriWcations subsequently yielded 200–350 g protein.
Silver gel analysis and Western blotting revealed the
purity of rCYR61 (Fig. 2). A major band of the expected
size (73 kDa) was visible in these analyses. The silver
staining reveals a very faint additional band of about
60 kDa (Fig. 2).
CAM assay
Since the CYR61 protein promotes angiogenesis in
vitro and in vivo [13,20,21,28] the chorioallantoic mem-
However, for CYR61 a direct proliferative action has
not been reported. Rather the protein added to human
Wbroblasts enhanced the proliferative response by bFGF
( D FGF-2) but was not proliferative by itself [12]. The
WST test was applied to primary human MSCs, EA hy
926 endothelial cells, and hFOB osteoblasts to further
prove the functionality of the protein, to analyze its
potential proliferative activity, and to identify additional
rCYR61 target cells within the bone microenvironment.
After 24 h of treatment with rCYR61 a marked induc-
tion of absorption ( D induction of proliferation) was
observed for all cell types (Fig. 4).
Discussion
CCN proteins play important roles in growth and
diVerentiation and are involved in cellular processes
such as migration, adhesion, and survival [1–3]. Since
CYR61 could play an integrative role in angiogenesis as
well as bone repair the recombinant protein would rep-
resent a suitable tool for elucidating its function within
the bone microenvironment.
Here, we expressed rCYR61 from baculovirus-
infected cells as a tool to analyze its function in cells of
the bone microenvironment. The established proangio-

Fig. 3. Proangiogenic activity of rCYR61 in the CAM assay. rCYR61 was added to the surface of the chorioallantoic membrane of fertilized eggs at
day 9 of hatching within a small silicone ring. Fifty nanogram (top row) or 1 g (bottom row) was used. (A) Pictures before addition of the protein,
and (B) (magniWcation 18£) and (C) (magniWcation 25£) represent pictures taken 4 days after the addition of rCYR61 to the membrane showing the
induction of blood vessel formation as is indicated by the branching of the membrane capillaries (see arrows).
 N. Schütze et al. / Protein Expression and PuriWcation 42 (2005) 219–225
Fig. 4. rCYR61 induces proliferation of MSC, hFOB cells, and endo-
thelial cells. Four thousand human bone marrow-derived MSC, osteo-
blasts (hFOB cells) or endothelial cells (EA hy 926-cell line) were
seeded per well of 96-well microplates and received serum-free
medium 1 day after seeding. Cells received 500 ng/ml rCYR61 for 24 h.
Subsequently, the WST-1 test as described in Materials and methods
was applied, the substrate was added for 4 h, and absorbance was mea-
sured in an ELISA reader at 450 nm.
genic role of CYR61 was used to prove the activity of
rCYR61 in the CAM assay. Additionally, a direct prolif-
erative response was observed on MSCs, osteoblasts,
and endothelial cells, thereby identifying target cells
within the bone microenvironment.
The expressed protein was highly pure according to
silver gel electrophoresis and Western blotting. Expres-
sion of CYR61 was also reported from the group of Lau
and Lam [5]. Whereas they used a His-tagged protein,
here an IgG-Fc tag was applied [29]. This strategy has
two advantages. First, the addition of the Fc-tag
enhances the solubility of the protein. CYR61 is known
223
to possess 10% cysteine residues and therefore represents
a very adhesive protein which could be easily lost during
puriWcation and handling. Also the protein tends to form
aggregates and to precipitate during or after puriWca-
tion. The addition of the Fc-tag minimizes these diYcul-
ties. A second reason to include the Fc-tag is the option
to use the fusion protein for detection of binding part-
ners. The protein can be easily immobilized on protein A
(or G) coupled carriers which then can be used with cel-
lular extracts (or puriWed candidates) for binding studies.
Although several binding partners have been identiWed,
additional binding proteins are likely to exist, since the
protein has complex actions on cells of bone, muscle,
vasculature, and the nervous system [1–3]. Integrin
receptors have been identiWed as binding partners in a
variety of cell types such as Wbroblasts, endothelial cells,
and platelets [3,5]. Recent reports indicate modulations
of pathways such as wnt-signaling in Xenopus laevis [30],
the  catenin c-myc p53 pathway in lung cancer cells
[31], and NF- B dependent apoptosis [32].
We cannot exclude the possibility that the Fc-tag of
the rCYR61 protein alters or inXuences the biochemical
characteristics of the protein. A native untagged protein
is not available for comparison in the functional tests, a
respective protocol is not established. However, the
CAM assay demonstrates that the known angiogenic
properties of the CYR61 protein are preserved in the
recombinant fusion protein. Generally, the Fc-tag
enhances the solubility of recombinant proteins. The fre-
quent use of Fc-tagged proteins in the literature, to our
knowledge, indicates the usability of these fusion pro-
teins for subsequent functional analysis. The established
role of CYR61 in angiogenesis relies on a mouse model
with functional inactivation [20] as well as additional in
vitro and in vivo data [13,21,32]. This proangiogenic
potential was used in this study to prove the activity of
the rCYR61 protein using the CAM assay.
Subsequently, the protein was used to elucidate its
function on cells of the bone microenvironment and to
identify additional target cells. CYR61 is considered to
promote proliferation. Here, we show that MSCs, endo-
thelial cells, and osteoblasts are target cells for CYR61
action, since they markedly responded to rCYR61 treat-
ment by an enhanced proliferation. To our knowledge,
this is the Wrst report that these cells directly respond to
CYR61. Previous data in the literature indicate the
enhanced proliferation of cells already stimulated by
growth promoting agents. Human Wbroblasts did not
show a proliferative response to treatment with CYR61,
but rather the proliferative eVect of bFGF was enhanced
[21]. Similarly, human umbilical vein endothelial cells
only responded with enhanced proliferation only due to
a cotreatment with VEGF plus CYR61 or VEGF alone,
not to the CYR61 treatment. Actions of CYR61 on pro-
liferation are complex and dependent on the cell type
studied.
224 N. Schütze et al. / Protein Expression and PuriWcation 42 (2005) 219–225
In addition to its angiogenic potential the CYR61
protein thus represents an integrator of various signaling
pathways which likely involves a multitude of interact-
ing proteins. Within the bone microenvironment osteo-
blasts, MSCs, and endothelial cells are target cells for
CYR61 action. In the future, the protein will be a valu-
able tool to elucidate its action on bone cells at the
molecular level with regard to binding partners as well as
gene expression patterns.
Acknowledgments
We are grateful to P. Schneider (University of Lau-
sanne, Switzerland) for providing an expression vector
containing the sequence of the Fc-tag. The hFOB-cell
line was kindly provided by S. Harris and T.C. Spelsberg
(Mayo Clinic, Rochester, USA). The EA hy 926 cells
were kindly provided by W. Baumgartner (University of
Würzburg, Germany). This work was supported by a
grant of the Deutsche Forschungsgemeinschaft to N.
Schütze and F. Jakob (KFO 103).
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