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Notes Synthesis and Characterization of Silklike Materials Containing the Calcium-Binding Sequence from Calbindin D9k or the Shell Nacreous Matrix Protein MSI60 Mingying Yang,† Tomoko Muto,† David Knight,‡ Andrew M. Collins,§ and Tetsuo Asakura*,† Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 Japan, Oxford Biomaterials Ltd., Units 14-15 Galaxy House, New Greenham Business Park, Thatcham, RG19 6HR, United Kingdom, and Department of Chemistry, Bristol University, Bristol BS8 1TS, United Kingdom Received June 13, 2007 Revised Manuscript Received September 15, 2007
Introduction Artificial bone- and dentinlike biomaterials for tissue regeneration are of considerable potential interest in clinical medicine. Bones and dentin are mainly composed of a combination of inorganic hydroxyapatite (HA) nanocrystals and the protein type I collagen. Hydroxyapatite formation is accelerated in vitro by certain specific extracellular matrix macromolecules acting as nucleators,1 while in vivo certain acidic noncollagenous proteins are thought to play an important role in hydroxyapatite deposition.2–4 Accordingly, many investigations are focused on artificial composite materials constructed from hydroxyapatite and proteins or synthetic polymers.5 Calbindin D9k is a small, acidic, and heat-stable protein found in the small intestine of all mammalian species.6,7 This protein is thought to be involved in fetal calcium uptake, uterine contractions, calcification of bone and teeth, and calcium transport and uptake in mammalian intestine.8 Calbindin D9k binds Ca2+ with a very high affinity (K ) 108 M-1). It belongs to the calmodulin superfamily of calcium-binding proteins characterized by the possession of a common helix–loop–helix motif, the EF-hand, which form high-affinity calcium-binding sites.9 MSI60 from the bivalve Pinctada fucata is one of the silklike matrix proteins present in the nacreous layer of mollusk shells where it is sandwiched between layers of aragonite, one of the crystalline forms of CaCO3.10–12 These matrix proteins regulate the epitaxial growth and thickness of the aragonite crystals and thus help to produce the extremely tough multilaminate structure of the shell nacre.13,14 The mechanical properties, biocompatibility, and resorbability of Bombyx mori fibroin, a predominantly β-sheet silk protein, give it considerable potential for use in strong and tough implantable biomaterials and scaffolds for tissue engineering.15 The fact that natural silk fibroins are capable of mineralization with the bone mineral hydroxyapatite suggests that they may * Corresponding author. E-mail:
[email protected]. Fax: (81)-42383-7733. † Department of Biotechnology, Tokyo University of Agriculture and Technology. ‡ Oxford Biomaterials Ltd. § Department of Chemistry, Bristol University.
have potential use as bone graft substitute materials.16,17 Our group previously designed a series of silklike peptides with the calcium-binding motif D9kL from Calbindin or that from Pinctada fucata MSI60 introduced between Ala-Gly or poly (Ala) repeating regions, the crystalline regions of natural silks.18,19 We found that the structure of the repeating regions can be controlled by the choice of both the organic solvents and the amino acid sequence so that they do not disrupt the secondary structure of D9kL and MSI60, suggesting that these motifs may retain their ability to bind calcium ions when flanked by silk-derived repeats.11,12 Furthermore we have shown that cast films prepared from silklike peptides containing the MSI60 domain are indeed able to bind calcium ions.19 In the present study, we report the design and production of two larger genetically engineered repetitive silklike proteins based on the two previously investigated18,19 nonrepetitive peptides. Our novel genetically engineered proteins each contain one of the two different calcium-binding sequences. The primary structure of these new proteins was [(AGSGAG)3AS(AGSGAG)3 ASEYDYDDDSDDDDEWD]2 (SM2) and [(AGSGAG)3 AS AAKEGDPNQLSKEE]8 (SC8). We show that SM2 but not SC8 inhibited calcium carbonate deposition from saturated solutions, demonstrating the former’s ability to bind calcium ions under the conditions used. We also show that SM2 but not SC8 can be dip-coated with hydroxyapatite.
Materials and Methods Production of Silklike Proteins. The general methods we used for the production of silklike proteins by genetic engineering are as described elsewhere.20,21 The oligonucleotide fragments encoding the crystalline region of B. mori silk fibroin block (S), Ca2+-binding sequence, N-terminal loop region of Calbindin D9k EF-hand domain (C), and Asp-rich region of Pinctada fucata MS160 (M) are shown in Figure 1. The DNA fragment for the crystalline region of B. mori silk fibroin block (S) and EF-hand domain of Calbindin D9k (C) were first inserted into BamHI-digested pUC118-linker (Takara Shuzo), Similarly, (S) and the Asp-rich region of Pinctada fucata MS160 (M) was inserted into BamHI and NheI-digested pUC118-linker. The DNA fragments of C and M were released on digestion of SpeI and NheI and ligated into SpeI and NheI-pUC118-linker-S to construct the monomer of silklike proteins SC and SM. Multimers of silklike proteins SC and SM were obtained by previously reported strategies involving head-to-tail ligation and orientation for NheI and SpeI sites.22 Multimerized DNA fragments encoding these recombinant proteins were inserted into BamHI- and HindIII-digested expression vector pET30a (Novagen). Expression vectors containing the verified genetic constructs were transfected into cells of the E.coli strain BL21(DE3)pLysS. Protein expression was induced by addition to the fermentor of IPTG to a final concentration of 1 mM at 25 °C. The recombinant protein was purified by affinity chromatography on a Ni-charged Hi-Trap column (GE Healthcare Bio-Sciences Corp.) The identity of the construct was confirmed by MALDI using an Applied Biosystems Voyager DE PRO. Initiation of Calcium Carbonate Precipitation. The effect of the recombinant silklike protein on calcium carbonate precipitation from its supersaturated solution was examined as described elsewhere.23,24 The rate formation of turbid calcium carbonate precipitate was followed over 7 min by monitoring the absorption at 570 nm of a solution prepared by adding 1.5 mL of 100 mM CaCl2 solution to a mixture of
10.1021/bm700665m CCC: $40.75 2008 American Chemical Society Published on Web 12/08/2007
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Figure 1. Oligonucleotide sequence used to produce the silklike proteins SC8 and SM2. (a) DNA sequence of crystalline region of Bombyx mori silk fibroin (S). (b) Oligonucletide sequence of N-terminal loop region of Calbindin D9k EF-hand domain (C). (c) Oligonucletide sequence of Asp-rich region of pinctada fucata MS160 (M).
Figure 2. (a) PAGE gel stained with ethidium bromide showing SpeI and NheI digestion analysis of SC8 oligonucleotides. Lane 1, PCR markers; lane 2, pUC118-monomer; lane 3, pUC118-dimer; lane 4, pUC118-tetramer; and lane 5, pUC118-octramer. (b) BamHI and NheI digestion analysis of SM2: Lane 1, 100 bp ladder (TOYOBO); lane 2, pUC118-monomer; lane 3, pUC118-linker-dimer.
1.5 mL of 100 mM NaHCO3 (pH 8.7) and 300 µL of SC8 or SM2 at initial concentrations ranging from 62.5 to 250 µg mL-1. The calcium carbonate precipitation experiment is very sensitive to pH. In general, the pH increases with time. Therefore we prepared fresh NaHCO3 solution (pH 8.7) repeatedly in order to maintain pH 8.7 carefully. In addition, to investigate the influence of the difference of silk motif on calcium binding, the previously synthesized model peptide (AG)6EYDY DDDSDDDDE-WD(AG)6,19 which is similar to one repetition of the SM2 sequence, was also used as a control. In all cases, the precipitated calcium carbonate formed did not start to sediment in the cuvette over the 7 min observation period, enabling absorption at 570 nm to be used as a valid measure of calcium carbonate formation Calcium Phosphate Deposition on Films. To prevent film from being soluble in calcium buffer or phosphate buffer, formic acid was used as solvent to prepare film from silklike protein as formic acid can induce β-sheet structure. The films of recombinant silklike protein were cast from 5% w/w SC8 and SM2 solutions in formic acid and air-dried at room temperature. During the casting, it is noted that the samples do not break down. One cycle of the dip-coating process was carried out on circular discs of film as follows: The film was first dipped at 37 °C in a 100 mM CaCl2 buffered with tris-(hydroxymethyl) ami-
nomethane (pH7.4) followed by a 60 mM Na2HPO4 solution. The film was then washed with distilled water and subsequently soaked in acetone to dry it This cycle was repeated up to 15 times to produce a heavy deposit of calcium phosphate on the film. A VE-7800 scanning electron microscopic (SEM) (KEYENCE) was used for survey purposes. This showed that 15 dip cycles gave heavy mineralization of SM2 but no detectable mineralization on SC8. For high-resolution SEM, SM2 films were sputter-coated with platinum to a thickness between 10 and 20 nm and examined in a JEOL JSM 6330 FEG SEM. Attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectra were obtained for mineralized SM2 using an FT-IR-4100 (JASCO). A Siemens D500 powder diffractometer with a Cu KR′ radiation source (λ ) 0.15405 nm) was used to demonstrate hydroxyapatite in the films. Samples were powdered before placing in a sample holder.
Results Gene Construction and Expression of Silklike Proteins. DNA electrophoresis of the SpeI- and NheI-digested cloning vectors (Figure 2a) confirmed the successful construction of pUC118-linker-SC monomer, dimer, tetramer, and octamer.
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Figure 3. Absorbance570 nm of solutions containing 1.5 mL of 100 mM NaHCO3 (pH 8.7) and 0.3 mL of protein solution (concentrations: see text) after addition of 1.5 mL of 100 mM CaCl2 (pH 8.7). (a) SC8, (b) SM2, and (c) model peptide (AG)6EYDYDDDSDDDDEWD(AG)6.19
Figure 4. High-resolution SEM images of calcium phosphate-coated SM2 film after 15 cycles of dip-coating. (a) Surface of the film showing a network formed from small platelike crystals. (b) Fractured film showing good adhesion of the mineral layer to the film. (c) FT-IR/ATR spectra of an SM2 film after 15 cycles of dip-coating.
Figure 2b shows an electrophoretogram of the DNA fragment of pUC118-linker-SM monomer and dimer, respectively. The DNA fragments released from pUC118-linker-SC8 and pUC118-
linker-SM2 were inserted into pET30a between BamHI and HindIII restriction sites, respectively. pET30a-SC8 and pET30aSM2 were transfected into BL21(DE3)pLysS cells, and the
Notes
encoded proteins were expressed using IPTG induction. The recombinant proteins were purified by affinity chromatography on a Ni-charged Hi-Trap column. The identity of the construct was confirmed by MALDI using an Applied Biosystems Voyager DE PRO. This showed a single high-molecular-weight peak at 18.2 kDa and at 32.7 kDa, respectively, which is in agreement with the theoretical MW of 17.6 kDa and 31.0 kDa for the His-Tagged SM2 and SC8, respectively. Evidence for the purification of the recombinant protein is summarized in Figures S1 and S2 in the Supporting Information. The yield of these silklike proteins was about 35 mg/L of the medium. Initiation of Calcium Carbonate Precipitation. The effect of the silklike proteins SC8 and SM2 on the inhibition of precipitation of calcium carbonate from pH buffered supersaturated solutions monitored spectrophotometrically is shown in Figure 3. The inhibition of precipitation of calcium carbonate can test calcium-binding ability of silklike proteins have SC8 and SM2, as indicated by a previous study.24 SC8 failed to inhibit calcium carbonate precipitation even at a final value of 125 µg/ ml (Figure 3a), while SM2 showed a dose-dependent inhibition of calcium carbonate precipitation over a concentration range of 62.5–125 µg/mL (Figure 3b). These observations indicate that SM2 binds calcium ions under the conditions used while SC8 does not. Furthermore, the model peptide (AG)6EYDYDD DSDDDDEWD(AG)6,19 which has a similar sequence to one repetition of SM2 [(AGSGAG)3AS(AGSGAG)3ASEY-DYDDDSDDDDEWD]2, also indicated dose-dependent inhibition of calcium carbonate precipitation (Figure 3c). This suggests the calcium binding is not affected by the length of silk motif, as SM2 has a similar ability to the model peptide (AG)6EYDYDDD SDDDDEWD(AG)6 and might be affected by selection of calcium-binding motif and interaction between functional motifs in the silklike protein. Calcium Phosphate Deposition on Films. Parts a–c of Figure 4 show the effect of dip-coating the films of SM2. The appearance of the coating was closely similar to that observed by Furuzono et al.25 after dip-coating silk fabric using a similar protocol. At high magnification, the mineral coat was seen to be composed of very small platelike crystals suggestive of hydroxyapatite (Figure 4a). Examination of the fractured surface of the film (Figure 4b) showed a relatively thick and continuous coating of mineral which did crack off the protein film, indicating that it adhered firmly. SEM examination of SC8 films showed no evidence of mineral on dip-coating. An FT-IR/ATR spectrum of a dip-coated film of SM2 (Figure 4c) with β-sheet conformation, judging from the amide I and II bands, showed peaks at 605 and 565 cm-1 assigned to PO43- v 4 bending and typical of an apatite. X-ray diffractometry of the same material gave two peaks that could be assigned to the 210 and 211 planes of hydroxyapatite (data not shown).
Discussion To investigate their potential for the production of mineralized composites, we designed and produced two genetically engineered proteins, SC8 and SM2, containing different calcium-binding motifs. SM2 inhibited calcium carbonate precipitation from supersaturated solutions, but SC8 did not indicating the former’s ability to bind calcium ions. The SM2 films repeatedly dipped in solutions containing Ca2+ followed by phosphate ions became coated with platelike crystals. FT-IR/ATR spectroscopy and X-ray diffractomery confirmed that the mineral crystals formed on SM2 were of hydroxyapatite. High-resolution SEM images indicated that the mineral coating rather firmly adhered to SM2 films. It is possible that the lack of mineralization of SC8 results from the very high affinity of its calcium-binding sites compared with
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those in SM2, causing the former artificial protein to bind tightly to the surface of the mineral, preventing further mineralization as suggested26 for other proteins inhibiting mineralization. Alternatively, the lower concentration of amino acid residues with negatively charged carboxyl side chains in SC8 may prevent mineral deposition. On the other hand, the lack of calcium binding of SC8 might be speculated the deformation of EF-hand that is the characteristic structure of Calbindin D9k in affinity calcium-binding sites.9 The calcium binding of SM2 can also be speculated as the ability of biospecific phenomena like ligand–receptor. Therefore, the three-dimensional structure of SC8 and SM2 in solution and solid states will be performed to dig out the factors’ effect on mineralization behaviors of these two proteins. Finally, our results indicate that artificial silk-based proteins based on SM2 may have potential for the development of mineralized coatings for orthopedic prostheses or mineralized composite materials for bone repair. Acknowledgment. T.A. acknowledges the support of Grantin-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan (18105007). D.P.K. and A.M.C. acknowledge support from the EU FP6 SilkBone project grant. Supporting Information Available. MALDI-TOFMS spectrum of His-tagged TS [(AGSGAG)3AS(AGSGAG)3ASEYDYDDDS DDDDEWD]2. MALDI-TOFMS spectrum of Histagged TS [(AGSGAG)3 AS AAKEGDPNQLSKEE]8. This material is available free of charge via the Internet at http:// pubs.acs.org.
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