Article pubs.acs.org/Langmuir
Silk−Silica Composites from Genetically Engineered Chimeric Proteins: Materials Properties Correlate with Silica Condensation Rate and Colloidal Stability of the Proteins in Aqueous Solution David J. Belton,† Aneta J. Mieszawska,‡ Heather A. Currie,†,‡ David L. Kaplan,‡ and Carole C. Perry*,† †
Biomolecular and Materials Interface Research Group, Interdisciplinary Biomedical Research Centre, School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham, UK NG11 8NS ‡ Department of Biomedical Engineering, Bioengineering and Biotechnology Center, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, United States S Supporting Information *
ABSTRACT: The aim of the study was to determine the extent and mechanism of influence on silica condensation that is presented by a range of known silicifying recombinant chimeras (R5: SSKKSGSYSGSKGSKRRIL; A1: SGSKGSKRRIL; and Si4−1: MSPHPHPRHHHT and repeats thereof) attached at the N-terminus end of a 15-mer repeat of the 32 amino acid consensus sequence of the major ampullate dragline Spindroin 1 (Masp1) Nephila clavipes spider silk sequence ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG]15X). The influence of the silk/chimera ratio was explored through the adjustment of the type and number of silicifying domains (denoted X above), and the results were compared with their non-chimeric counterparts and the silk from Bombyx mori. The effect of pH (3−9) on reactivity was also explored. Optimum conditions for rate and control of silica deposition were determined, and the solution properties of the silks were explored to determine their mode(s) of action. For the silica−silk−chimera materials formed there is a relationship between the solution properties of the chimeric proteins (ability to carry charge), the pH of reaction, and the solid state materials that are generated. The region of colloidal instability correlates with the pH range observed for morphological control and coincides with the pH range for the highest silica condensation rates. With this information it should be possible to predict how chimeric or chemically modified proteins will affect structure and morphology of materials produced under controlled conditions and extend the range of composite materials for a wide spectrum of uses in the biomedical and technology fields.
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INTRODUCTION The use of alien materials in the human body has increased dramatically over the past 50 years although the origin of their use dates at least to the time of the pharaohs who used natural silk for sutures and more recently 150 years ago when amalgam dental fillings were first used.1 Over the years these materials have become more sophisticated with biocompatibility and bioacceptance being important. Silks provide a valuable natural source of biomaterials that can be fabricated into differing physical forms including fibers, films, and gels for physiological applications for controlled release2−4 and as tissue engineering constructs.5−9 In addition to being well tolerated biologically,10−12 silk has many physical properties which are highly valued such as its elasticity and tensile strength.13−15 In the past 20 years there has been growing interest in understanding the properties of silks with a view to generating new materials based on spider silks and bioengineered variants of silkworm and spider silks.16 Although spiders produce a wide range of silks17 having different functions such as protection for the lining the egg case (aciniform silk), some are more glue-like (pyriform and aggregate silks) while others have a more structural role such as the major and minor ampullate silks used in the web © 2012 American Chemical Society
and draglines; none have been commercialized due to the difficulty of domestication of the cannibalistic Araneae. Silks have impressive mechanical properties of strength and toughness that rivals those of synthetic high performance fibers such as Kevlar;18 however, in compression none of the silks perform well as fibres or in highly porous gel networks as under these conditions they are less resistant to compressive deformation. Amorphous silica is an inorganic material which is well accepted biologically19 and is found in nature as various silicate minerals and in its soluble form, SiOH4 (orthosilicic acid, also sometimes called monosilicic acid), in all water sources at concentrations ranging from a few parts per million (ppm) up to ∼100 ppm. It is from this source that many organisms are able to sequester silica to form structural materials. Diatoms, for example, form elaborate shells (thecae) which provide structure and protection while still allowing the diffusion of nutrients and gases. The controlled deposition of silica in organisms such as diatoms involves a number of proteins and shorter peptides Received: December 23, 2011 Revised: January 31, 2012 Published: February 7, 2012 4373
dx.doi.org/10.1021/la205084z | Langmuir 2012, 28, 4373−4381
Langmuir
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that include post-translationally modified silaffins.20 These have been shown to promote silica formation in vitro. As an example, Silaffin 1A (a 19 amino acid peptide containing polyamine modified lysine groups) has been shown to regulate silica deposition and morphology from pre hydrolyzed tetraalkoxysilane solutions in vitro,21 a property also shared with its synthetic nonmodified counterpart, R5 (SSKKSGSYSGSKGSKRRIL).22 In addition the same study found that fragments of the R5 peptide such as the 11-mer, SGSKGSKRRIL (A1), had similar effects. Other unrelated peptides with silicifying ability have also been identified by screening methods (phage display) to isolate peptide sequences which show binding affinity to silica surfaces. One example, MSPHPHPRHHHT (Si4−1 or A3),23 showed the greatest activity in terms of the level of silica deposition from preprepared orthosilicic acid solutions when compared with a number of other silica binding phage peptide sequences. Silica is itself a brittle material with poor tensile properties, but it is hard and can be used in composite materials to complement the inherent properties of other components. The possibility of forming silk scaffolds with controllable porosities using silica to impart structural rigidity could potentially provide biomedical science with a range of slowly biodegradable implantable materials that also have the capacity for doping with growth factors, nutrients, or targeted drugs to be released during the lifetime of the implant.24 In principle, silk−silica composites could be tailored using the bio-inspired silicification process performed in vitro at circum-neutral pH and ambient temperature. In our earlier studies using the 32 amino acid consensus sequence of the major ampullate dragline Spindroin 1 (Masp1) Nephila clavipes spider silk sequence ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG]n) and a silica binding peptide (R5: SSKKSGSYSGSKGSKRRIL)), we have shown that protein chimeras can be generated using genetic engineering and further demonstrated the positive effect of the binding peptide on the silicifying properties of silk.25 We have more recently shown that these materials promote osteoblast development with the upregulation of key markers associated with bone formation.26 To further advance the potential application of such spider silk−silica composite materials for applications including those in biomedical science, knowledge of the reaction environment available for silica formation is needed together with an understanding of the compatibility of silica formation with maintenance of the excellent structural characteristics of the silk itself such as its microcrystalline β-sheet structure which confers some of its impressive mechanical properties. In this contribution we therefore consider the effect of the identity of the peptide used on silica formation and on the structure of the silk component itself. The activity of peptides (R5: SSKKSGSYSGSKGSKRRIL), a truncated form (A1: SGSKGSKRRIL), and a peptide identified by Phage display (Si4−1 or A3: MSPHPHPRHHHT), as well as multiples of this sequence as A3(2) and A3(3), were compared to controls: a silk 15-mer without a specific silicifying domain and a native silk fibroin from Bombyx mori. The effect of pH (3−9) on composite formation (particle size and size distribution) and protein secondary structure for the silk proteins associated with the siliceous materials are compared. Optimum conditions for rate and controlled silica morphology deposition were determined, and the solution properties of the silks were explored to determine their mode(s) of action. The data show that knowledge of the solution behavior of the chimeras together with an understanding of silica formation can be used to predict the likely compatibility of the two phases.
Article
MATERIALS AND METHODS
Recombinant Silk and Chimeras. The 15-mer repeat of the consensus sequence (major ampullate spidroin: MaSp I) from the spider dragline silk (Nephila clavipes) (SGRGGLGGQGAGAAAAAGGA-GQGGYGGLGSQGT)(15)X where C-terminal domains, X, of A1 (SGSKGSKRRIL-15-mer A1), A3(1),(2),(3) (MSPHPHPRHHHT(1−3)-15-mer A3(1−3)), R5 (SSKKSGSYSGSKGSKRRIL-15mer R5) or absent for the non-chimera-(15-mer) were prepared using cloning strategies previously described.25,27 All of the constructs were His tagged for the purpose of purification. Bombyx mori Silk Fibroin Preparation. The silk materials were prepared using our previously reported procedures.28 Briefly, silk cocoons were degummed using 0.5% sodium carbonate solutions and water washing and the separated silk fibers dried in air. The silk fibroin was regenerated by dissolving in lithium bromide solution (9 M) to ∼5% protein concentration, dialysing rapidly (
