Self-assembled adhesive biomaterials formed by a genetically designed fusion protein

Article abstract of DOI:10.1039/c8cc07475e

Here we report a recombinant protein (MS) obtained by genetic fusion of a mussel foot protein (Mfp3) motif into a silk spidroin (MaSp1). The MS not only self-assembled into a supramolecular fibre, as does the parent MaSp1, but also showed enhanced adhesiveness resulting from the DOPA-containing Mfp3 portion. The successful incorporation of the wet adhesiveness of Mfp3 into the well-structured assembly of MaSp1 may provide a new insight for the genetic design of underwater adhesive recombinant proteins by utilizing the structural features of a spidroin protein.

Full text of DOI:10.1039/c8cc07475e

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Self-assembled adhesive biomaterials formed by a
genetically designed fusion protein†
Cite this: Chem. Commun., 2018,
54, 12642
Pulakesh Aich,‡a Jaeyeon An,‡b Byeongseon Yang,‡c Young Ho Ko,a
a
James Murray,a Hyung Joon Cha, *c Joon Ho Roh,*a
Received 14th September 2018,
Accepted 18th October 2018
Junghyun Kim,
Kyeng Min Park *a and Kimoon Kim
*
ab
DOI: 10.1039/c8cc07475e
rsc.li/chemcomm
Here we report a recombinant protein (MS) obtained by genetic fusion glue proteins.6 Development of biomaterials with such a collec-
of a mussel foot protein (Mfp3) motif into a silk spidroin (MaSp1). The tion of properties is extremely challenging.
MS not only self-assembled into a supramolecular fibre, as does the
Mussel foot proteins (Mfp1, 2, 3, 4, 5 and 6) present in the
parent MaSp1, but also showed enhanced adhesiveness resulting from surface adhered flatten plaques of mussel byssus have been
the DOPA-containing Mfp3 portion. The successful incorporation of extensively studied to understand their remarkable underwater
the wet adhesiveness of Mfp3 into the well-structured assembly of adhesion properties.7,8 Among these, Mfp3 and Mfp5 have been
MaSp1 may provide a new insight for the genetic design of underwater found to be important proteins for underwater interfacial adhesion
adhesive recombinant proteins by utilizing the structural features of a because of their relatively large proportion (20–30 mol% in the
spidroin protein.
proteins) of L-3,4-dihydroxyphenylalanine (DOPA) compared to that
of other Mfps.9 Numerous studies revealed that DOPA is an
Nature exhibits many biomaterials with remarkable chemical important chemical moiety in Mfps for underwater adhesion to
and physical properties that often inspire researchers to develop various organic/inorganic surfaces. Until now, the reported roles of
new functional materials. For example, tough biomimetic fibres DOPA include the dehydration of a wet surface, and adhesion
inspired by spider silk draglines have outstanding mechanical to the surface through various interactions including hydrogen
properties;1,2 such properties are attributed to their well-defined bonding, metal oxide coordination and cation–p interactions.10
tertiary structure which is self-assembled from major ampullate To take advantage of this interesting behaviour, DOPA has been
spidroin 1 (MaSp1, accounting for the crystalline fraction of chemically conjugated to polymers, nanoclays and metal nano-
spider webs).3,4 The large proportion of nonpolar and hydro- particles as well as enzymatically transformed on proteins from
phobic amino acids such as glycine and alanine plays a crucial tyrosine, and exploited as a crosslinker and adhesive to develop
role in forming a b-sheet structure by providing robust inter- and various soft-materials including adhesive hydrogels, actuators
intra-molecular interactions. The assembly uses its good elastic and self-healing materials.11–18 Although these materials
properties to maintain its structure; it achieves this by releasing showed the great potential of Mfps as adhesive biomaterials,
applied stress through stick-slip deformation of the protein the adhesion property of the materials was not as good as that
assemblies.5 To capture prey in its web, natural spider silks are of the natural Mfps which are hierarchically organized with
not only mechanically robust and flexible but also adhesive. The other biomolecules in plagues. Since the adhesion property was
combined properties are a result of the hierarchical organiza- significantly influenced by the structural order of the materials,19
tion of various silk proteins including silk dragline, spiral and a rational design of bioadhesives which complements a well-
ordered structure to adhesive Mfps is highly required to enhance
the adhesion ability.
Using a genetic engineering, herein, we demonstrate a
a Center for Self-assembly and Complexity, Institute for Basic Science (IBS), Pohang,
successful genetic fusion of Mfp3 (from Mytilus galloprovincialis)20
37673, Republic of Korea. E-mail: jhroh@ibs.re.kr, kmpark@ibs.re.kr,
showing adhesiveness to MaSp1 (from Nephila clavipes)21 self-
kkim@postech.ac.kr; Web: http://csc.ibs.re.kr
b Department of Chemistry, Pohang University of Science and Technology, Pohang,
assembled into a well-defined supramolecular structure to develop
a new type of recombinant protein-based adhesive biomaterials
(Fig. 1). The structural characteristics of the newly designed
recombinant protein (MS) resembled those of MaSp1; namely, they
formed fibres. Furthermore, it showed enhanced adhesiveness in a
wet condition compared to that of MaSp1, which suggests the great
37673, Republic of Korea
c Department of Chemical Engineering, Pohang University of Science and Technology,
Pohang, Korea. E-mail: hjcha@postech.ac.kr; Web: http://magic.postech.ac.kr
† Electronic supplementary information (ESI) available: Supplementary figures
and table, experimental procedures. See DOI: 10.1039/c8cc07475e
‡ These authors contributed equally to this work.
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Fig. 2 (a) SDS-PAGE gel of the purified recombinant proteins stained with
Coomassie brilliant blue and (b) western blotting of (a) with anti His-tag
primary antibody which was visualized by HRP conjugated-secondary
antibody.
of formation of the blue-coloured diformazan from NBT upon
oxidation of DOPA in a solution of NBT/K-glycinate. In the NBT
assay with MS, we observed a blue-coloured band when blotted
the protein solution on a nitrocellulose membrane, indicating
that MS has DOPA converted from tyrosine (Fig. S2, see the
Fig. 1 Schematic illustration of genetic fusion of Mfp3 into MaSp1 and
their self-assembly to form DOPA containing supramolecular fibres.
potential of this new genetic design for recombinant proteins as ESI†). The quantitative analysis of the UV-Visible absorption
advanced bio-adhesives. revealed that ca. 3.2 tyrosine residues out of 16 (20%) had been
For expression of the recombinant protein (27.9 kDa), plasmid converted to DOPA in MS. In addition, MALDI-ToF MS analysis
DNA containing Mfp3 (6.8 kDa) and MaSp1 (22.0 kDa) was used; showed 50 Da higher molecular ion peak of MS after treating
the proteins were connected by a flexible glycine-serine linker with tyrosinase compared to that of MS before treating
‘‘GGGGS’’ for enhanced functionally active protein expression.22 (Fig. S1c, see the ESI†). It supported transformation of ca. 3.1
Hexa-histidine tag (His-tag) was inserted at the N-terminus of the DOPA from tyrosine residues which is well-matched with that
recombinant protein for immobilized metal affinity chromato- from UV-Vis. The structure of MS was analysed by circular
graphy (IMAC) purification23 (Table S1 for the individual sequence dichroism (CD) and SEM. CD spectra of Mfp3, MaSp1 and MS
for the proteins, see the ESI†). The target protein in a lysate of the revealed that the shape of the spectrum of MS is different from
bacteria was anchored to Ni-nitrilotriacetic acid (Ni-NTA) resins that of Mfp3, but highly similar to that of MaSp1 (Fig. 3a).
having His-tag binding affinity. Since DOPA is a non-canonical These CD spectra suggested that the MaSp1 portion in MS
amino acid, the protein anchored to the resin was treated with an governed the final structure of the protein assembly. To visua-
enzyme (tyrosinase) for transformation of tyrosine to DOPA.24,25 lize the structure, we performed SEM of the proteins after
After His-tag affinity chromatography, the purified protein was lyophilisation (see the ESI†). The SEM images of these proteins
analysed by SDS-PAGE gel (See the ESI† for synthesis and purifica- showed that MS formed fibres (diameter = 770 ꢀ 90 nm,
tion). The protein band stained with Coomassie brilliant blue Fig. 2b) similar to those of MaSp1 (diameter = 750 ꢀ 40 nm,
appeared at 28 kDa (Fig. 2a), which agreed well with sum of Fig. 2d) whereas Mfp3 did not form such fibres (Fig. 2c). In
molecular weight of Mfp3 (6.8 kDa) and MaSp1 (22.0 kDa). The addition, MS with tyrosine before transformed into DOPA also
mass of the protein was confirmed by a molecular ion peak at showed a similar fibre formation with MaSp1 in SEM (Fig. S3, see
27.9 kDa (Fig. S1a, see the ESI†) in matrix assisted laser desorption the ESI†). These results also strongly suggested that the MaSp1
and ionization time of flight mass spectroscopy (MALDI-ToF MS). portion played a crucial role in forming such a hierarchical
In addition, the detection of the purified proteins in a western blot supramolecular structure of the recombinant protein, MS. MaSp1
assay by anti His-tag primary antibody with horseradish peroxidase and Mfp3 in MS seemed to act as a rigid and flexible segment,
(HRP)-conjugated secondary antibody (Fig. 2b) confirmed the respectively, in the protein to self-assemble into a fibre, similar to
successful expression and purification of the target recombinant those in synthetic macromolecules forming unique self-assembled
protein, MS.
To validate the incorporation of DOPA into MS, we per-
structures with functions.27–29
To study the influence of the hierarchical structure of MS on
formed a nitroblue tetrazolium (NBT) assay (see the ESI†).26 its elasticity, we performed dynamic mechanical analysis of MS
This assay quantifies the amount of DOPA in the protein by (see the ESI†). MS (10 mL, 2.5 nmol) was loaded in between two
detecting increase of absorption at 530 nm, which is a measure circular mica surfaces before the measurement (see the ESI†).
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Fig. 3 (a) CD spectra of MS (solid line), Mfp3 (small dashed line) and MaSp1 (large dashed line), and SEM images of (b) MS, (c) Mfp3 and (d) MaSp1.
Fig. 4 Dynamic mechanical analysis of MS, MaSp1 and Mfp3 using a
rheometer. Shear strain dependent elastic modulus (G0) and loss modulus
(G00) at constant frequency sweep (10 rad sꢁ1).
The MS was found to have a typical viscoelastic soft material
behaviour showing non-linear shear strain-dependent loss of elastic
modulus (G0) which was also higher than the loss modulus (G00)
over the whole range of the shear strain (Fig. 4). Comparison of the
modulus values (G0 and G00) for Mfp3 and MaSp1, measured in the
same condition as above (Fig. 4), revealed that MaSp1 had at least Fig. 5 (a) Schematic illustration of SFA analysis of surface adhesion
interactions and (b) the result of SFA measurement for Mfp3, MaSp1 and
MS. The normalized forces (F/R) and its corresponding interaction energies
10-fold higher elastic moduli than Mfp3. This result clearly sup-
ported that MS formed well-organized fibres making it significantly
more elastic than Mfp3. The elasticity of MS is closer to that of
per unit area (defined as F/1.5pR) are shown on the left and right ordinates,
respectively.
MaSp1 than it is to Mfp3, suggesting that the elasticity of MS
seemed to be originated from the MaSp1 motif of MS. A close
comparison of MS and MaSp1 revealed that MS was ca. 2 times (force/radius, F/R) to detach MS from mica was measured to be
more elastic than MaSp1. This result indicated that the genetic 55 mN mꢁ1 (Fig. 5b), which was converted to 12 mJ mꢁ2 as surface
fusion of Mfp3 reinforced elasticity of the self-assembled structure adhesion energy per unit area (W) by following the equation,
of MS. The enhanced elasticity via the long-range self-assembly into W = F/1.5pR, as reported earlier.30 We also performed the same
fibres reduces failure not only in cohesion but also in adhesion experiment as above with MaSp1, instead of MS, to directly
across the adhesive/surface interface because they are better able to compare the surface adhesion energy between them. This measure-
dissipate energy through their flexible structure.
ment revealed that MaSp1 also showed adhesion ability with the
After the structural and the dynamic mechanical analysis of the energy of 4 mJ mꢁ2 (Fig. 5b) which is 3-fold lower than that of MS.
recombinant protein-based supramolecular structures, we investi- This result clearly indicated that MS showed significantly enhanced
gated the surface adhesiveness of MS to mica in a wet condition. surface adhesion ability compared to MaSp1 which does not
The interaction force between MS and mica was measured using contain a DOPA moiety in the SFA measurement. When compared
Surface Force Apparatus (SFA) as per the literature report30 (see the to the SFA result of Mfp3 (14 mJ mꢁ2), MS showed 1.2-fold less
ESI†). One mica surface was treated with a sufficient amount of the adhesion energy. At a glance, this result seemed to indicate that MS
protein to cover the whole surface, then washed and contacted to did not provide enhanced surface adhesion property compared to
the other mica surface for 10 min (Fig. 5a). The normalized force Mfp3. However, considering the density of the MS molecules on the
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surface in the SFA measurements estimated based on the hydro- Notes and references
dynamic radii of the proteins (1.9 nm and 3.3 nm for Mfp3 and MS,
1 M. Xu and R. V. Lewis, Proc. Natl. Acad. Sci. U. S. A., 1990, 87,
7120–7124.
2 F. Vollrath, J. Biotechnol., 2000, 74, 67–83.
3 L. Romer and T. Scheibel, Prion, 2008, 2, 154–161.
4 X. Hu, K. Vasanthavada, K. Kohler, S. McNary, A. M. Moore and
C. A. Vierra, Cell. Mol. Life Sci., 2006, 63, 1986–1999.
5 S. W. Cranford, A. Tarakanova, N. M. Pugno and M. J. Buehler,
Nature, 2012, 482, 72–76.
respectively), measured by diffusion-ordered spectroscopy (DOSY)
in D2O (Fig. S4, see ESI† for detail), the number of MS molecules
can be 2.9-fold fewer than that of Mfp3. The adhesion energy per
protein molecule of MS is 2.5-fold greater than that of Mfp3. In
other words, one protein molecule of MS can be 2.5-fold more
adhesive than that of Mfp3.
6 T. Scheibel, Microb. Cell Fact., 2004, 3, 14.
Taken together, a recombinant protein of MS containing Mfp3
and MaSp1 motifs showed significantly enhanced adhesiveness
compared to MaSp1 alone. Such enhanced underwater adhesiveness
of MS seems to be due to the molecular basis of the recombinant
protein. Specifically, the genetic fusion of Mfp3 into MaSp1 yields
more elastic and underwater sticky self-assembled fibres with
presentation of DOPA moieties. Higher elasticity induced by the
formation of supramolecular structures probably plays a central
role in alleviating both cohesive and adhesive stress of MS by
better dissipating energy across protein molecules.
7 B. P. Lee, P. B. Messersmith, J. N. Israelachvili and J. H. Waite, Annu.
Rev. Mater. Res., 2011, 41, 99–132.
8 A. H. Hofman, I. A. van Hees, J. Yang and M. Kamperman, Adv.
Mater., 2018, 30, 1704640.
9 Q. Lin, D. Gourdon, C. Sun, N. Holten-Andersen, T. H. Anderson,
J. H. Waite and J. N. Israelachvili, Proc. Natl. Acad. Sci. U. S. A., 2007,
104, 3782–3786.
10 G. P. Maier, M. V. Rapp, J. H. Waite, J. N. Israelachvili and A. Butler,
Science, 2015, 349, 628–632.
11 M. R. Kim and T. G. Park, J. Controlled Release, 2002, 80, 69–77.
12 M. Brodie, L. Vollenweider, J. L. Murphy, F. Xu, A. Lyman, W. D. Lew
and B. P. Lee, Biomed. Mater., 2011, 6, 015014.
13 A. K. Gaharwar, S. A. Dammu, J. M. Canter, C. J. Wu and G. Schmidt,
Biomacromolecules, 2011, 12, 1641–1650.
In summary, we demonstrated a recombinant protein-based
bio-adhesive by inserting one natural protein into another,
namely Mfp3 into MaSp1. The resulting spidroin-based protein
forming an elastic fibril structure showed underwater adhesiveness
because of the mussel foot protein motif. The enhanced properties
were confirmed by dynamic mechanical analysis with a rheometer
and underwater adhesion measurement by SFA. In principle, the
number of DOPA moieties in the protein can be increased
by enhancing the conversion of DOPA from tyrosine in the
protein with adopting longer glycine-serine linkers in genetic
design or in vivo residue-specific incorporation of DOPA using
misaminoacylation;31 thus the adhesiveness of the biomaterials
can be further improved. This study may provide new insights
for the design of underwater adhesive recombinant proteins
based on the structural features of a spidroin protein, which
can be potentially useful for various bioapplications including
biomedical uses such as glues for soft tissues.
14 B. P. Lee and S. Konst, Adv. Mater., 2014, 26, 3415–3419.
15 B. K. Ahn, D. W. Lee, J. N. Israelachvili and J. H. Waite, Nat. Mater.,
2014, 13, 867–872.
16 L. Han, X. Lu, K. Liu, K. Wang, L. Fang, L. T. Weng, H. Zhang,
Y. Tang, F. Ren, C. Zhao, G. Sun, R. Liang and Z. Li, ACS Nano, 2017,
11, 2561–2574.
17 Q. Zhu and Q. Pan, ACS Nano, 2014, 8, 1402–1409.
18 J. Horsch, P. Wilke, M. Pretzler, M. Seuss, I. Melnyk, D. Remmler,
A. Fery, A. Rompel and H. G. Borner, Angew. Chem., Int. Ed., 2018,
DOI: 10.1002/anie.201809587.
19 R. J. Stewart, T. C. Ransom and V. Hlady, J. Polym. Sci., Part B: Polym.
Phys., 2011, 49, 757–771.
20 S. C. Warner and J. H. Waite, Mar. Biol., 1999, 134, 729–734.
21 O. Tokareva, M. Jacobsen, M. Buehler, J. Wong and D. L. Kaplan,
Acta Biomater., 2014, 10, 1612–1626.
22 R. Trinh, B. Gurbaxani, S. L. Morrison and M. Seyfzadeh, Mol.
Immunol., 2004, 40, 717–722.
23 P. N. Hengen, Trends Biochem. Sci., 1995, 20, 285–286.
24 C. Zhong, T. Gurry, A. A. Cheng, J. Downey, Z. T. Deng, C. M. Stultz
and T. K. Lu, Nat. Nanotechnol., 2014, 9, 858–866.
25 M. J. C. Long and L. Hedstrom, ChemBioChem, 2012, 13, 1818–1825.
26 P. B. Issopoulos, Pharm. Weekbl., 1989, 11, 213–217.
27 Y. Kim, W. Li, S. Shin and M. Lee, Acc. Chem. Res., 2013, 46,
2888–2897.
28 Z. Huang, S. K. Kang, M. Banno, T. Yamaguchi, D. Lee, C. Seok,
E. Yashima and M. Lee, Science, 2012, 337, 1521–1526.
29 B. Sun, Y. Kim, Y. Q. Wang, H. X. Wang, J. Kim, X. Liu and M. Lee,
Nat. Mater., 2018, 17, 599–604.
This work was supported by the Institute for Basic Science (IBS)
[IBS-R007-D1] (to K. K.) and Marine Biotechnology Program of the
Korea Institute of Marine Science and Technology Promotion
funded by the Ministry of Oceans and Fisheries (to H. J. C.).
30 B. Yang, C. Lim, D. S. Hwang and H. J. Cha, Chem. Mater., 2016, 28,
7982–7989.
31 B. Yang, N. Ayyadurai, H. Yun, Y. S. Choi, B. H. Hwang, J. Huang,
Q. Lu, H. Zeng and H. J. Cha, Angew. Chem., Int. Ed., 2014, 53,
13360–13364.
Conflicts of interest
There are no conflicts to declare.
This journal is ©The Royal Society of Chemistry 2018
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