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 J_ID: ZA1 Customer A_ID: 06-0385 Cadmus Art: JBMA31348 Date: 4-APRIL-07 Stage: I Page: 1

Silklike materials constructed from sequences

of Bombyx mori silk fibroin, fibronectin, and elastin

Mingying Yang,1 Chikako Tanaka,1 Kazuo Yamauchi,1 Kosuke Ohgo,1 Masato Kurokawa,2 Tetsuo Asakura1
1
Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan
2
Sanyo Chemical Industries, Higashiyama-Ku, Kyoto 605-0995, Japan

Received 8 June 2006; revised 29 November 2006; accepted 9 January 2007

Published online 00 Month 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31348

Abstract: Two silklike proteins, [TGRGDSPAGG(GAGA
GS)3AS]5 (FS5) and [TGRGDSPA-(GVPGV)2GG(GAGA
GS)3AS]8 (FES8) were designed to demonstrate the superior
performance as biomaterials of silklike proteins. The former
protein consists of the crystalline domain sequence,
(GAGAGS)n from Bombyx mori silk fibroin and cell-adhesive
sequence TGRGDSPA coming from fibronectin-containing
RGD triplet. The additional sequence (GVPGV)n from elastin
was included in the latter protein. The considerably higher
cell-adhesion activities of these proteins for NHDF and
VERO cells were observed by comparing with those of silk-
like materials without RGD sequences and also the crystal-
line fraction of B. mori silk fibroin. This tendency was inde-
pendent of the treatments, 4.5M LiClO4 or formic acid (FA),
on silklike proteins. Their activities are also higher than those
of commercial Fibronectin FTM for NHDF cell. Their struc-
tural characterization was studied using 13C solid-state
NMR. Although the overlapped peaks in usual 13C CP/MAS
NMR spectra make the detailed structural analysis difficult,
the methyl resonance regions observed using dipolar
dephasing NMR were very useful for the analysis. The pres-
ence of both random coil and b-sheet structures was
observed in these proteins clearly. The content of b-sheet
structure in both proteins increases after FA treatment when
compared with the lyophilized samples. The production of
electrospun nanofibers from their hexafluoroacetone solution
was also tried. The silklike protein FES8 could prepare
nonwoven silk fibers although FS5 could not. Ó 2007 Wiley AQ2
Periodicals, Inc. J Biomed Mater Res 81A: 000–000, 2007
Key words: silklike protein; fibronection; elastin; cell adhe-
sion; solid-state NMR; electrospinning

INTRODUCTION biodegradability, silk fibroin can address the me-

Tissue engineering provides an alternative to tradi-
tional treatment protocols by replacing living tissue
with tissue grown in vitro, that is, in turn, designed
and engineered to meet the needs of each individual
patient and repair site.1 This approach uses tissue-
specific cells that are grown on a scaffolding material
to give a three-dimensional structure with the goal
of creating a functional organ or tissue. Accordingly,
the scaffold should be designed by mimicking the
structure and biological function of native extracellu-
lar matrix (ECM) proteins, which provide mechani-
cal support and regulate cell activities.
Because of many inherent superior properties as
biomaterials, in the viewpoint of mechanical proper-
ties, environmental stability, biocompatibility, and
Correspondence to: T. Asakura; e-mail: asakura@cc.tuat.
ac.jp
Contract grant sponsor: Ministry of Education, Science,
Culture, and Sports of Japan; contract grant number:
18105007
' 2007 Wiley Periodicals, Inc.
chanical and functional requirements of material
options in the field of tissue engineering.2,3 For
example, the silk fibroin fiber spun from domestic
silkworm, Bombyx mori, has been used commercially
for biomedical sutures for decades. The successful
attachment and growth of fibroblasts on silk fibroin
films prepared from B. mori silk fibroin have been
reported.4 Silk decorated with the cell-adhesive
sequence Arg-Gly-Asp (RGD) derived from fibronec-
tin has been used for a 3D scaffold for bone forma-
tion.5,6 Cappello et al.7–9 have first developed silklike
protein with cell-adhesive activity by the introduc-
tion of amino acid sequence containing (GAGAGS)9
from B. mori silk fibroin crystallized in b-sheet con-
formation and RGD present in a turn, enabling bind-
ing to RGD-adhesion receptors on the cell surface.
This silklike protein has been available for commer-
cial use with the name of Pronectin FTM.10 In con-
trast to silk fibroin from B. mori, domestic silkworm,
we explored a novel silklike protein with higher cell
adhesive and growth activities based on silk fibroin
from Samia cynthia ricini, wild silkworm. The pri-
mary structure of this silklike protein was designed

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 J_ID: ZA1 Customer A_ID: 06-0385 Cadmus Art: JBMA31348 Date: 4-APRIL-07
2 YANG ET AL.
as [DGG(A)12GGAASTGRGDSPAAS]5 by combining
the sequence RGD with polyalanine (Ala)12, the crys-
talline region of S.c.ricini silk fibroin.11 We also
found production of interferon-b by NB1-RGD fibro-
blast cells cultured on protein and peptide mem-
branes prepared from silk fibroin, model peptides of
silk fibroin [(AG)n] containing RGD peptide.12 Thus,
it has been proved that silklike proteins containing
both the sequences of RGD and crystal region of silk
fibroin have high cell adhesive and growth activities.
Cappello’s group has also constructed a family of
silk–elastinlike block copolymers that are varied in
copolymer composition and sequence using genetic
engineering techniques for controlled drug deliv-
ery.13 The periodic incorporation of VPGVG, which
is derived from the mammalian protein elastin, was
expected to promote an increase in the flexibility of
silklike proteins.10 Solid state NMR spectra14–17 of
elastin model peptide, (VPGVG)6, and silklike pro-
tein, [(GVPGV)2GG(GAGAGS)3AS]n, confirmed that
the incorporation of elastinlike blocks, VPGVG, into
silk fibroin sequences increases a conformational dis-
tribution locally, which is expected to give additional
functionalities such as flexibility and elasticity to the
properties of silk fibroin. These additional properties
seem useful in the development of biomaterials
based on silk fibroin. The nonwoven fabrics are
among the most promising material forms used in
various tissue-engineering applications, because their
high specific surface area and highly porous three-
dimensional structures are desirable for high-density
cell and tissue cultures.18 Furthermore, some research
groups explored the nonwoven electrospun nano-
fiber from B. mori silk fibroin and proved to be a
useful new scaffolding biomaterial applicable for a
wide range of target tissues.19–23
In this study, we will attempt to develop a new
scaffolding biomaterial by increasing the cell-adhe-
sive activity of currently available silklike proteins
and improving the processing ability of these silklike
proteins into nonwoven form by focusing on revi-
sion of primary structure. For this purpose, the
following two silklike proteins were designed. One
of these is designed as [TGRGDSPAGG(GAGAGS)3
AS]n, whose primary structure contains the three
repeats of (GAGAGS) and the cell-adhesive sequence
TGRGDSPAG derived from fibronectin. The other
silklike protein has the primary structure, [TGRGDSPA
(GVPGV)2GG(GAGAGS)3AS]n, where the elastin
sequence (GVPGV)2 was incorporated between the
sequences TGRGDSPA and (GAGAGS)3. The gene
construction, expression, and purification of these
two silklike proteins were performed. The cell-adhe-
sion assay for NHDF and VERO cells was performed
by comparing these two silklike proteins with previ-
ously synthesized silklike proteins without RGD
sequences together with crystalline fraction of silk
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
ID: srinivasanv Date: 4/4/07 Time: 16:41
Stage: I Page: 2
fibroin. Prior to cell-adhesion experiments, these silk-
like proteins were treated by 4.5M LiClO4 or formic
acid (FA). Here, the former treatment is the same as
reported for commercial Fibronectin FTM24 and their
activities are compared among them. The cell-growth
assay was also by using VERO cell.
Their secondary structure of these two proteins AQ3
prepared as lyophilized samples and samples by FA
treatment was studied using both 13C CP/MAS
NMR and dipolar dephasing NMR. Especially, the
methyl resonance regions observed in the dipolar
dephasing NMR spectra were very useful in the
structural analysis through the determination of the
fraction of both random coil and b-sheet structures.
The production of electrospun nanofibers from their
hexafluoroacetone (HFA) solution was also tried for
these two silklike proteins to examine their capabil-
ity as scaffolding materials in nonwoven silk form.
MATERIALS AND METHODS
Materials
E. coli strain DH5a was used for the propagation and
construction of plasmids, and E. coli strain BL21(DE3)-
pLysS was used for the production of proteins. Synthe-
sized DNA fragments with phosphorylation were pur-
chased from Sigma Genosys, Japan. Restriction enzymes
and ligase were purchased from Takara Shuzo. Plasmid-
pUC118 and pET30a were obtained from Takara Shuzo
and Novagen, respectively. VERO, African green monkey
kidney, and NHDF, normal human dermal fibroblasts,
were obtained from Dainippon Pharmaceutical, Japan. All
other chemicals were of analytical grade. Bacteria were
grown in an enriched medium, and DNA manipulations
and transformation were performed as described by Sam-
brook et al.25 DNA sequencing was performed on an ABI
PRISMTM 377 Auto-sequencer according to the user’s man-
ual. E. coli cell density was measured at k ¼ 600 nm on a
Hitachi U-3200 spectrophotometer in quartz cuvettes with
a path length of 1 cm. Batch cultures were performed on
MD-6C Fermentor (B. E. Marubishi) with a 1.2-L vessel.
Gene construction
The duplex DNA shown in Figure 1(a) was ligated into F1
pUC118 to construct pUC118-linker vector, which provides
SpeI and NheI restriction enzyme sites for the polymeriza-
tion of target DNA. The oligonucleotide fragments encod-
ing the crystalline domain of B. mori silk fibroin block (S)
and the elastic motif block (E) were illustrated in Figure
1(b,c), respectively. The oligonucleotide fragments [Fig.
1(dI,dII)], which encoded the cell-adhesive sequence
derived from fibronectin (F), were arranged as SpeI and
BamHI [Fig. 1(dI)] and EcoRI and flat [Fig. 1(dII)] restric-
tion enzyme sites at 50 - and 30 -terminals, respectively, to
control the sequence orientation of monomer through
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 J_ID: ZA1 Customer A_ID: 06-0385 Cadmus Art: JBMA31348 Date: 4-APRIL-07
SILKLIKE MATERIALS CONSTRUCTED FROM SEQUENCES
Figure 1. The designed oligonucleotide sequences for
silklike proteins FS5 and FES8. (a) DNA sequence of the
synthetic adapter inserted into pUC118 to create the
pUC118-linker, (b) oligonucleotide sequences of the B. mori
silk fibroin block, (c) oligonucleotide sequences of the
elastin block, and (d) oligonucleotide sequences of the cell-
adhesion block (I was inserted into silklike proteins FES8
and II into silklike proteins FS5).
ligation sites. Therefore, the monomer of silklike protein
FS was constructed by ligating the oligonucleotide S and F
in Figure 1(dII) and inserted into the EcoRI and BamHI-
digested pUC118. The construction of monomer of silklike
protein FES was completed through the following two
steps. In the first step, the oligonucleotides S and E were
ligated and then inserted into the EcoRI and BamHI-
digested pUC118. The second step was performed by
inserting the oligonucleotides F in Figure 1(dI) into the
SpeI and BamHI-digested pUC118-SE. The multimers of
silklike proteins FS and FES were obtained by using previ-
ously reported strategies involving the head-to-tail ligation
and orientation for NheI and SpeI sites.26 Multimerized
DNA fragments encoding these two recombinant proteins
were inserted into the BamH I and HindIII-digested expres-
sion vector pET30a.
Protein expression and purification
Expression vectors containing the verified genetic con-
structs were transformed into the E. coli strain BL21(DE3)-
pLysS and subsequent cultures were grown in Luria-
Bertain broth (TB) containing chloramphenicol (25 lL/mL)
and kanamycin (25 lL/mL). Batch cultures of volumes of
1 L were allowed to grow at 378C to an optical density
between 0.8 and 1.5, as determined by optical absorbance
measured at 600 nm (OD600). Protein expression, under the
control of a bacteriophage T7 promoter, was induced by
the addition of IPTG to a final concentration of 1 mM.
Expression was continued for 1–4 h before harvest via
centrifugation (8500 rpm, 40 min, 48C). The cells were
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Stage: I Page: 3
3 AQ1
collected and stored at
708C before purification. Protein
expression and the level of expression were confirmed by
Western-blot analyses, where a T7-tag antibody-horseradish
peroxidase conjugate was used for visualization. Frozen
cells were thawed on ice, and resuspended in the lysis
buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole,
pH 8.0) at 5 mL/g wet weight, and cells were disrupted
by supersonication. Cellular debris was removed by
centrifugation (14,500 rpm, 30 min, 48C). To purify the
fusion protein by using His-tags, the supernatant was
loaded onto a nickel chelate affinity column, which was
charged with Niþ2. The column was lysed with lysis buffer
and washed with a wash buffer (50 mM NaH2PO4,
300 mM NaCl, 20 mM imidazole, pH 8.0). The protein was
eluted with an elution buffer (50 mM NaH2PO4, 300 mM
NaCl, 250 mM imidazole, pH 8.0). The eluted protein
was dialyzed against distilled water for 3 days and then
lyophilized.
13
C solid state NMR measurement
The lyophilized FS5 and FBS8 powders and also the
samples after their FA treatment were used for 13C solid
state NMR experiments. About 35 mg of the sample was
contained in a cylindrical rotor with outer diameter of
4 mm. 13C CP/MAS NMR spectra were acquired on a
Chemagnetics CMX-400 spectrometer operating at 100 MHz,
with a CP contact time of 1.5 ms, TPPM (two-pulse phase-
modulated) decoupling, and magic angle spinning at
5 kHz. Spectra of 10,000–20,000 scans were accumulated
over a spectral width of 80 kHz, with a recycle delay of
0.5 s. Chemical shifts were reported in parts per million
relative to TMS as a reference. To determine a possible
change of the secondary structure of these proteins, methyl
13
C NMR peaks were selectively examined by dipolar
dephasing experiments of 13C solid state NMR.27 This
experiment is a variation of the CP experiment, in which a
delay time is introduced after the contact time to suppress
methine and methylene peaks, with the dephasing time of
60 ls. Deconvolution of the peaks observed at the
region,10–30 ppm in the dipolar dephasing NMR spectra,
was performed by referring the conformation-dependent
13
C chemical shifts of Ala residue; *16.0–16.2 ppm for
random coil and *19.8–20.9 ppm for b-sheet and by
assuming Gaussian line shape. Although the peak position
is the same between random coil and silk I in the Ala Cb
peak of silk fibroins, the broadened peaks at 16.0–
16.2 ppm could be ascribed to random coil rather than silk
I. However, determination of the secondary structure of
FES8 was performed by subtracting the contribution of
the Val Cg peak to the Ala Cb peak in the region of 10–
30 ppm.
Cell-adhesion assay
NHDF and VERO cells were used for the cell-adhesion
assay. The solution with concentration of 100 lg/mL was
prepared by dissolving silklike proteins FS5 and FES8 in
4.5M LiClO4 aqueous solution or FA, and was added to
96-well microplates, 16 lL/well. Here, the former solution
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
 J_ID: ZA1 Customer A_ID: 06-0385 Cadmus Art: JBMA31348 Date: 4-APRIL-07
4 YANG ET AL.
was used for cell-adhesion experiment of commercial
Fibronectin F.24 The plates were desiccated under vacuum
for 2 h after drying at room temperature for 12 h. The
wells were washed with physiological saline twice, and
then, saturated with 0.2% BSA for 2 h. Before adding
100-lL DMEM medium, the wells were washed twice with
physiological saline. The plates were kept in a 378C incu-
bator for 1 h. Cultured cells were washed with PBS, de-
tached with 0.25% trypsin, washed, and suspended again
to *1.0 3 106 cells/mL in DMEM. This cell suspension
was added to wells, which were kept in incubator, so that
the number of cells was 1.0 3 104 cells/well, and incu-
bated for 60 min at 378C, 5% CO2. After the supernatants
were removed, the number of cells was counted using
Tetracolor OneTM, Seikagaku, Japan. For a comparison,
Pronectin F, the crystalline fraction of B. mori silk fibroin,
and two silklike proteins without RGD sequence, [GGAGS-
GYGGGYGHGYGSDGG(GAGAGS)3]428 and [(GVPGV)2GG
(GAGAG-S)3AS]8,14 previously prepared in our laboratory,
were also used for cell-adhesion assay. Each experiment
was repeated eight times and the averaged values together
with the standard deviation were calculated relative to the
value for the experiment with nontreated plate. Significant
level of silklike proteins compared to nontreatment was
evaluated by statistical analysis, t test. The statistical sig-
nificance in the t test and p value was calculated and was
shown in the figure legends.
Cell-growth assay
VERO cells were also used for the cell-growth assay.
Cell-growth plates were prepared as same as cell-adhesion
assay, until the plates were desiccated under vacuum
condition. After washing with physiological saline twice,
100-lL VP-SFM medium was added and the plates were
kept in a 378C incubator for 1 h. Cultured cells were
washed with PBS, detached with 0.25% trypsin, washed,
and resuspended to *1.0 3 106 cells/mL in VP-SFM. This
cell suspension was added to wells, which were kept in an
incubator, so that the number of cells was 2.0 3 103 cells/
well, and incubated for 3 days at 378C, 5% CO2. After the
removal of supernatants, the number of cells was also
counted using Tetracolor One. Each experiment was
repeated eight times and the averaged values together
with the standard deviation were calculated relative to the
value for the experiment with nontreated plate. Significant
level of silklike proteins compared to nontreatment was
also calculated by t test.
Electrospinning of silklike proteins
We reported previously the preparation of nonwoven
silk fibroin fibers from the hexafuoroactetone solutions of
several silks including wild silks using electrospinning
method.19 The preparation method for the two silklike pro-
teins in this article is essentially the same as the previous
one. The silklike proteins were dissolved in HFA and the
concentrations were in the range from 6 to 20 wt %. In the
electrospinning process, a high electric potential was
applied to a droplet of silk solution at the tip (0.45 mm in
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internal diameter) of a plastic capillary. The electrospun
fiber was collected on the mesh, which was placed at dis-
tance of 10–15 cm from the tip of the plastic capillary.
Here, aluminum sheet was used as mesh. A voltage of 15–
30 kV was applied to the wire in the capillary by a high
voltage power supply to inject the silk solution on the
aluminum sheet.
SEM observation
Scanning electron microscope (KEYENCE VE-7800,
Japan) was used to measure the fiber diameters and distri-
bution of diameters. The alumni sheet, which collected the
nonwoven fiber, was cut into shape of 3 cm 3 3 cm. The
fiber diameter distribution of the nonwoven fibers was
sampled from 100 positions of fiber-crossing in the SEM
pictures.
RESULTS
Construction and expression of silklike proteins
Protein expression efforts involved use of the com-
mercially available expression vector pET30a, which
contains [His]6 sequence at the N-terminus and C-
terminus of the silklike proteins for purification by
immobilized metal affinity chromatography. The
plasmid vectors encoded silklike proteins FS5 and
FES8 were transformed into BL21(DE3)pLysS and
the encoded proteins were expressed upon induction
of IPTG. Proteins were purified from the Ni-NTA
affinity chromatography under native conditions.
Figure 2 shows the SDS-page gel stained with Coo- F2
massie Brilliant Blue R-250 for the purified His-
tagged silklike proteins FS5 and FES8. The purity of
the silklike proteins was checked by SDS-page in
each single band. The molecular weight of FS5 and
FES8 was estimated from the SDS-page band as
about 22.0 and 33.0 kDa, respectively, which is in
agreement with the theoretical prediction of the mo-
lecular weights. The yields of FS5 and FES8 were
40.8 and 20.5 mg/L, respectively.
Structural characterization of silklike proteins
Solid-state NMR methods were used to determine
the B. mori silk fibroin structures: type II b-turn of
silk I29 and heterogeneous structures of silk II,30 con-
formational transition from silk I to silk II for B. mori
silk fibroin, or the model peptide (AG)15 by FA treat-
ment based on isotropic 13C chemical shift. 13C CP/
MAS NMR spectra show the silk I pattern when
these samples were dissolved in 9M LiBr and then
dialyzed against water. After FA treatment of these
samples, the 13C CP/MAS NMR spectra changed
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SILKLIKE MATERIALS CONSTRUCTED FROM SEQUENCES 5 AQ1

taking into account of the conformation-dependent

13
C chemical shifts of Ala residue; *16.0–16.2 ppm
for random coil and *19.8–20.9 ppm for the b-sheet.
Although the peak position is very close to each
other between random coil and silk I in the Ala Cb
peak of silk fibroins, the broadened peak of the
17 ppm indicating the structure is random coil rather
than silk I. Only Ala Cb peak was observed for silk-
like protein FS5 [Fig. 4(a,b)]. The lyophilized powder
of silklike protein FS5 adopts predominantly random
coil (80%) with small amount of b-sheet (20%). After
FA treatment, the structure was changed largely;
predominantly b-sheet (70%) and 30% random coil.

Figure 2. SDS-page analyses of purified proteins. Lane 1,

protein markers; lane 2, silklike protein FS5; lane 3, silklike
protein FES8.

largely, indicating the silk II structure.30,31 This

change is clearly observed when Ala Cb region of
the spectra was carefully examined. The sequence
(GAGSGA)3 is included in the primary sequences of
both silklike proteins FS5 and FES8, and therefore,
the FA treatments of silklike proteins are expected to
induce the structural change. The 13C CP/MAS
NMR spectra of these silklike proteins were illus-
F3 trated in Figure 3. Clearly, structural change occurs
for FS5 by FA treatment; the narrow spectral pattern
was observed for FA-treated preparation as viewed
from the region 10–70 ppm [Fig. 3(a,b)]. On the other
hand, there appears only small change for FES8 after
FA treatment [Fig. 3(c,d)]. However, it seems diffi-
cult to discuss the structure and structural change
because of severe peak overlapping. Therefore,
another NMR technique, the dipolar dephasing
NMR, was used here. As shown in Figure 3(e), only
methyl peaks remain in the 13C solid state NMR
spectra as a result of suppressed methine and meth-
ylene peaks with dephasing time, 60 ls was used.27
The methyl regions in the dipolar dephasing NMR Figure 3. 13C CP/MAS NMR spectra of (a) silklike pro-
tein FS5 after dialysis treatment; (b) FS5 after FA treatment;
F4 experiments were summarized in Figure 4 together (c) silklike protein FES8 after dialysis treatment; (d) FES8
with the deconvolution peaks. The deconvolution after FA treatment; (e) dipolar dephasing NMR spectrum
was performed by assuming Gaussian line shape, of FES8 after FA treatment.

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6 YANG ET AL.

Thus, the large conformational change of FS5 was

observed by FA treatment. On the other hand, in the
case of silklike protein FES8, the Ala Cb peak is
overlapped with the signal of Val Cg methyl peak
from the elastin sequence (GVPGV)2, and therefore,
elimination of the Val Cg peak is necessary for
discussing the structure and structural change of the
Ala Cb peak. Figure 4(c,e) shows the deconvoluted
Ala Cb and Val Cg carbons of silklike protein FES8.
The peak at 19.0 ppm assigned to the Val Cg carbon
should be included in the 13C NMR spectrum and
the chemical shift of the Val Cg carbons is independ-
ent of the treatments as reported previously.17 There
are eight Ala residues and four Val residues in one
repeat of the primary sequence of silklike protein
FES8: [TGRGDSPA(GVPGV)2GG(GAGAGS)3AS], and
thus, the fraction of both Ala Cb and Val Cg peaks
are expected to be 50%. Therefore, the Val Cg peak
with fraction of 50% was subtracted from the peak
in the region of 10–30 ppm in the dipolar dephasing
NMR spectra [Fig.4(c,e)]. After subtraction of 50%
Val Cg peak in Figure 4(c,e), the difference spectra
are shown in Figure 4(d,f). As shown in Figure 4(d),
random coil (80%) and b-sheet (20%) were deter-
mined for the lyophilized powder. The structural
change after FA treatment is relatively small for
FES8 as evaluated from 80 to 60% (FA treatment).
The chemical shifts and the fraction of b-sheet and
random coil were summarized in Table I. T1

Cell adhesion and growth activities

of silklike proteins

The cell adhesion and growth activities of silklike

proteins FS5 and FES8 were measured together with
the commercialized Pronectin F, another two re-
combinant silklike proteins, which have the primary
structure of [GGAGSGYGGGYGHGYGSDGG(GAGA

TABLE I
13
The C Chemical Shiftsa and The Fraction of b-Sheet
and Random Coil of Ala Residues in Silklike
Proteins FES8 and FS5 Determined from Ala C b
Peak of Dipolar dephasing NMR
Sample Chemical Fraction
Figure 4. The spectral deconvolution of the peaks at Shift (ppm) (%)
10–30 ppm region in the dipolar dephasing NMR spectra
FS5 after dialysis treatment 20.5 20
by assuming Gaussian line shape. (a) The Ala Cb spectrum
16.0 80
of lyophilized silklike protein FS5 powder; (b) and that of
FS5 after FA treatment 19.8 70
FS5 after FA treatment; (c) the Ala Cb and Val Cg spectra
16.0 30
of lyophilized silklike protein FES8 powder; (d) and its cor-
FES5 after dialysis treatment 20.9 20b
responding Ala Cb spectra after subtraction of the Val Cg
16.0 80b
peak; (e) the Ala Cb and Val Cg spectra of FES8 after FA
FES5 after FA treatment 21.0 40b
treatment; (f) the corresponding Ala Cb spectra after sub-
16.2 60b
traction of the Val Cg peak. Dashed lines (



) indi-
a
cated the Ala Cb spectra. Dot-dashed lines (


) Parts per million from TMS.
b
indicated the Val Cg peak. The fraction was calculated from decomposed peaks
after substracting Val C g peek at 10—30 ppm (Fig. 4).

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SILKLIKE MATERIALS CONSTRUCTED FROM SEQUENCES 7 AQ1

Figure 5. The histograms of the cell-adhesive assays of (I) silklike protein FS5, (II ) silklike proteins FES8, (III) Pronectin
F, (IV) recombinant silklike protein [GGAGSGYGGGYGHGYGSDGG(GAGAGS)3]4, (V) recombinant silklike protein
[(GVPGV)2GG(GAGAGS)3AS]8, (VI) CP fraction, and (VII) nontreatment. The NHDF cell was used (a) means the samples
treated with LiClO4 and (b) treated with FA. Each value represents the mean of eight separate determinations 6 SD. (*p <
0.01, **p < 0.5).

GS)3]428 and [(GVPGV)2GG(GAGAGS)3-AS]8,14 and
the crystalline fraction of B. mori silk fibroin were
used for the comparison. The experiments of cell-ad-
hesion activity with NHDF and VERO cells are sum-
marized, relative to the case of nontreatment plate
F5
F6 (100%) as histograms as shown in Figures 5 and 6,
respectively. Here, the results were analyzed accord-
ing to the statistical treatment, t test. When compared
with the cases of silklike proteins, [GGAGSGYGG-
GYGHGYGSDGG(GAGAGS)3]4 and [(GVPGV)2GG
(GAGAGS)3AS]8, and the crystalline fraction, which
has no RGD sequences, it turned out that silklike
proteins FS5, FES8, and Pronectin F-containing RGD
sequence after LiClO4 or FA treatments have consid-
erably higher cell-adhesion activities (p < 0.01 or p <
0.5 for NHDF cells, and p < 0.005 or p < 0.025 for
VERO cells ) as shown in Figures 5 and 6. In Figure 5,
FS5 and FES8 after LiClO4 treatment show higher
cell-adhesion activity than these samples after FA
treatment for NHDF cells. In contrast, for VERO
cells, silklike protein FS5 after FA treatment shows
higher cell-adhesion activity than those after LiClO4
treatment (Fig. 6). The silklike proteins FS5 and FES8
have also higher cell-adhesion activities in response
to VERO cells than to NHDF cells (Figs. 5 and 6).
The cell-growth activities of silklike proteins FS5
and FES8 were also measured using the VERO cells
(Fig. 7). In both cases of LiClO4 and FA treatments, F7
silklike proteins, FS5 and FES8, and Pronectin F
show higher cell-growth activities than other sam-
ples (p < 0.5 or p < 0.3). The difference in the cell-
growth activity among these silklike proteins and
Pronectin F become smaller compared with the case
of the cell-adhesion activity. Thus, the high cell
adhesion and cell-growth activities of silklike pro-
teins FS5 and FES8 as well as Pronectin F are mainly
due to the presence of RGD sequence in their pri-
mary structure.

Figure 6. The histograms of the cell-adhesive assays of (I) silklike protein FS5, (II) silklike protein FES8, (III) pronectin F,
(IV) recombinant silklike protein [GGAGSGYGGGYGHGYGSDGG(GAGAGS)3]4, (V) recombinant silklike protein
[(GVPGV)2GG(GAGAGS)3AS]8, (VI)CP fraction, and (VII) nontreatment. The VERO cell was used (a) means the samples
treated with LiClO4 and (b) treated with FA. Each value represents the mean of eight separate determinations 6 SD. (*p <
0.005, **p < 0.025).

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8 YANG ET AL.

Figure 7. The histograms of the cell-growth assays of (I) silklike protein FS5, (II ) silklike proteins FES8, (III) pronectin F,
(IV) recombinant silklike protein [GGAGSGYGGGYGHGYGSDGG(GAGAGS)3]4, (V) recombinant silklike protein
[(GVPGV)2GG(GAGAGS)3AS]8, (VI)CP fraction, and (VII) nontreatment. The VERO cell was used (a) means the samples
treated with LiClO4 and (b) treated with FA. Each value represents the mean of eight separate determinations 6 SD. (*p <
0.3, **p < 0.5).

Electrospinning of silklike protein increase in the concentration to 10 wt % [Fig. 9(e)].

To process silklike proteins FS5 and FES8, nonwo-
ven fiber was prepared from the HFA solutions
using electrospinning method.19,32 At first, the con-
centration of the dope for FS5 was optimized for the
preparation of nonwoven fiber using electrospinning.
More than the concentration 8 wt %, the HFA solu-
F8 tion became too viscous and could not spin. Figure 8
shows the SEM morphology of the nonwoven fibers
spun from the HFA solutions of FS5 with the concen-
trations of 6 and 8%. Only beads and beaded fibers
were obtained with the concentrations of 6 and 8%,
respectively. In contrast, the continuous fiber was
generated for FES8 with the concentrations of 8, 10,
and 12 wt % using electrospinning method as shown
F9 in Figure 9. The diameter distribution of the fibers is
also shown. The averaged fiber diameter is the
smallest and the fiber diameter distribution is the
narrowest for the case of 8 wt % [Fig.9(d)]. The mean
fiber diameter was increased to about 200 nm with
In the case of 12 wt %, the diameter becomes thick
and distribution tends to be broad with arrangement
from 200 to 400 nm [Fig. 9(f)]. The mean fiber diame-
ter increases with the increase in concentration,
meaning that the diameter can be controlled by
changing the concentration of the dope. Thus, silk-
like protein FES8 is superior to silklike protein FS5 as
view from the manner of preparation of nonwoven
silk fiber. This might be because of the incorporation
of elastin sequence (GVPGV)2, which improves the
elasticity and solubility of silklike protein.
DISUSSION
To develop a new scaffolding biomaterial, two
silklike proteins FS5 and FES8, were designed and
produced by genetical engineering. The characteriza-
tion of these two silklike proteins was performed by
structural analysis with 13C solid state NMR, assay

Figure 8. Scanning electron micrograph of beads and beaded fibers prepared from silklike proteins FS5 in HFA solutions
with the concentrations of (a) 6 and (b) 8 wt % using electrospinning method.

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SILKLIKE MATERIALS CONSTRUCTED FROM SEQUENCES
Figure 9. Scanning electron micrograph of nonwoven fibers prepared from the silklike proteins FES8 in HFA solutions
with the concentrations of (a) 8, (b) 10, and (c) 12 wt % using electrospinning method. The distributions of the diameter of
the fibers (d), (e), and (f) are respectively shown.
of cell-adhesive activity, and formation of nonwoven
fiber. As is well-known, genetic engineering to con-
struct, clone, and express native and synthetic genes
encoding recombinant proteins is a powerful method
for modifying a variety of properties, through appro-
priate choice of different units, the number of unit in
each multimer, the spacing between them, and the
number of repeats of multimer combination assem-
bly. Thus, by varying the number and arrangement
of primary monomers, a variety of silklike materials
with different physical and chemical properties can
be obtained. The silklike protein FS5 is designed
as [TGRGDSPAGG(GAGAGS)3AS]5 whose primary
structure contains five repetitions of alternate arran-
gement of (GAGAGS)3 and cell-adhesive sequence
TGRGDSPAG derived from fibronectin, while silk-
like protein FES8 has the primary structure, [TGR-
GDSPA(GVPGV)2GG(GAGAGS)3AS]8, where the elas-
tin sequence (GVPGV)2 was incorporated between
the sequences of TGRGDSPA and (GAGAGS)3. The
incorporation of elastin sequence (GVPGV)2 between
the sequences of TGRGDSPA and (GAGAGS)3 results
in that silklike protein FES8 shows different charac-
ters from those of silklike protein FE5. As is shown
in Figure 4, silklike protein FS5 undergoes remark-
able structural transition from random coil to b-sheet
by FA treatment, which has been observed in native
B. mori silk fibroin. However, FES8 prepared by FA
treatment adopts predominantly random coil confor-
mation. Thus, introduction of elastin sequence
(GVPGV)2 changes the structure of FE5 significantly.
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9 AQ1
Furthermore, silklike proteins FS5 and FES8 as well
as Pronectin F show high cell-adhesion activity after
LiClO4 and FA treatments because of the introduc-
tion of RGD sequence in their primary structure. In
particular, for NHDF cells, synthesized silklike pro-
teins FS5 and FES8 have clearly higher cell-adhesion
activities than that of Pronectin F (Fig. 5). This might
be due to the difference in their primary structure
and the manner of interaction between cells and pro-
teins. In addition, it was found that silklike protein
FS5 cannot be processed to nonwoven fiber while
silklike protein FES8 can be done due to the incorpo-
ration of elastin sequence (GVPGV)2.
As summarized from the results of structural
analysis, assay of cell-adhesive activity and forma-
tion of nonwoven fiber of silklike proteins FS5 and
FES8, it was found that subtle change in the primary
structure of proteins can change the character of
proteins remarkably. This work is seen as a first step
towards designing high performance synthetic silks
for use in tissue engineering. Further research is
underway to address the relationship between
sequence and structure and properties and develop-
ment of potentially useful materials.
CONCLUSIONS
For the development of silklike materials for tissue
engineering, two silklike proteins FS5 and FES8 were
designed and prepared. The primary structure of
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10
silklike proteins FS5 is constructed from the crystal-
line region of B. mori silk fibroin (GGAGS)3 and cell-
adhesive sequence RGD derived from fibronectin,
while that of silklike proteins FES8 is composed with
(GGAGS)3, elastin sequence (GVPGV)2, and the
sequence RGD. Both proteins have considerably
higher cell adhesion and growth activities for NHDF
and VERO cells when compared with the case of the
crystalline fraction of B. mori silk fibroin. The latter
silklike protein is suitable for the preparation of non-
woven silk fiber using electrospinning method and
thus, this can be used for the materials of the tissue
engineering such as wound dressing.
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 J_ID: ZA1 Customer A_ID: 06-0385 Cadmus Art: JBMA31348 Date: 4-APRIL-07 Stage: I Page: 11

AQ1: Kindly check whether the short title is OK as given.

AQ2: Per scientific convention, symbols/characters representing genes are set in italic while the same are
set normally (in roman) when they represent proteins. Kindly indicate with an underline (in the proofs)
all symbols/characters that represent genes.

AQ3: Something seems to be missing here. Kindly check.AQ4: Kindly provide the article title for Refer-
ence 9.

ID: srinivasanv Date: 4/4/07 Time: 16:42 Path: J:/Production/JBMM/Vol00000/070159/3B2/C2JBMM070159