Silk-silica composites from genetically engineered chimeric proteins: materials properties correlate with silica condensation rate and colloidal stability of the proteins in aqueous solution.

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](15)X). 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.

Bioinspired silicification of silica-binding peptide-silk protein chimeras: comparison of chemically and genetically produced proteins.

Novel protein chimeras constituted of "silk" and a silica-binding peptide (KSLSRHDHIHHH) were synthesized by genetic or chemical approaches and their influence on silica-silk based chimera composite formation evaluated. Genetic chimeras were constructed from 6 or 15 repeats of the 32 amino acid consensus sequence of Nephila clavipes spider silk ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG](n)) to which one silica binding peptide was fused at the N terminus. For the chemical chimera, 28 equiv of the silica binding peptide were chemically coupled to natural Bombyx mori silk after modification of tyrosine groups by diazonium coupling and EDC/NHS activation of all acid groups. After silica formation under mild, biomaterial-compatible conditions, the effect of peptide addition on the properties of the silk and chimeric silk-silica composite materials was explored. The composite biomaterial properties could be related to the extent of silica condensation and to the higher number of silica binding sites in the chemical chimera as compared with the genetically derived variants. In all cases, the structure of the protein/chimera in solution dictated the type of composite structure that formed with the silica deposition process having little effect on the secondary structural composition of the silk-based materials. Similarly to our study of genetic silk based chimeras containing the R5 peptide (SSKKSGSYSGSKGSKRRIL), the role of the chimeras (genetic and chemical) used in the present study resided more in aggregation and scaffolding than in the catalysis of condensation. The variables of peptide identity, silk construct (number of consensus repeats or silk source), and approach to synthesis (genetic or chemical) can be used to "tune" the properties of the composite materials formed and is a general approach that can be used to prepare a range of materials for biomedical and sensor-based applications.

Silica in plants: biological, biochemical and chemical studies.

BACKGROUND: The incorporation of silica within the plant cell wall has been well documented by botanists and materials scientists; however, the means by which plants are able to transport silicon and control its polymerization, together with the roles of silica in situ, are not fully understood. RECENT PROGRESS: Recent studies into the mechanisms by which silicification proceeds have identified the following: an energy-dependent Si transporter; Si as a biologically active element triggering natural defence mechanisms; and the means by which abiotic toxicities are alleviated by silica. A full understanding of silica formation in vivo still requires an elucidation of the role played by the environment in which silica formation occurs. Results from in-vitro studies of the effects of cell-wall components associated with polymerized silica on mineral formation illustrate the interactions occurring between the biomolecules and silica, and the effects their presence has on the mineralized structures so formed. SCOPE: This Botanical Briefing describes the uptake, storage and function of Si, and discusses the role biomolecules play when incorporated into model systems of silica polymerization as well as future directions for research in this field.

Resolution of complex monosaccharide mixtures from plant cell wall isolates by high pH anion exchange chromatography.

The use of high pH anion exchange chromatography combined with pulsed amperometric detection has been established as an effective and sensitive method for the separation, detection and quantification of monosaccharides from a wide range of sources. However, careful examination of the separation conditions required is necessary to ensure that a complete monosaccharide profile can be determined from structures such as the plant cell wall which is a complex network of both neutral and charged polysaccharides. This study has investigated the optimal conditions required for the analysis of such a challenging mixture, including both the stationary and mobile phase minimising co-elution and reducing method complexity. The preferred methods have been used to successfully identify and quantify the monosaccharide components of a selected extract from the plant cell wall of the primitive higher plant Equisetum arvense.

Chemical evidence for intrinsic 'Si' within Equisetum cell walls.

This contribution provides the first detailed chemical evidence for the association of 'Si' with soluble carbohydrate and proteinaceous components of Equisetum arvense cell walls. For Equisetum telmateia and E. arvense, the presence of intrasilica organics trapped within phytolithic silica deposits and organics packaged within silica coated particles on the outside of spores have previously been identified. The current paper shows that 'Si' is also found intimately associated with cell wall polymers that can be released using mild extraction procedures (CDTA and Na(2)CO(3)/NaBH(4)) that do not solubilise the mineral phase. The isolates comprise both protein and carbohydrate components with increases in 'Si' (up to slightly more than 1% by weight) being particularly linked to increased levels of protein within the extracts. The general composition of the cell wall isolates associated with 'Si' was very different to that previously found for intrasilica and spore related material with much lower levels of charged amino acids, particularly basic amino acids being detected. The range of monosaccharides detected was much wider than for the other silicified materials investigated. It is possible that 'Si' in some form could act to crosslink the cell wall polymers thereby providing a modest improvement in rigidity/stability of the cell wall against biotic and abiotic stresses. This study demonstrates that distinct differences are to be found between extra- and intrasilica organics in the cell wall of E. arvense.

Bioengineered silk protein-based gene delivery systems.

Silk proteins self-assemble into mechanically robust material structures that are also biodegradable and non-cytotoxic, suggesting utility for gene delivery. Since silk proteins can also be tailored in terms of chemistry, molecular weight and other design features via genetic engineering, further control of this system for gene delivery can be considered. In the present study, silk-based block copolymers were bioengineered with poly(L-lysine) domains for gene delivery. Ionic complexes of these silk-polylysine based block copolymers with plasmid DNA (pDNA) were prepared for gene delivery to human embryonic kidney (HEK) cells. The material systems were characterized by agarose gel electrophoresis, atomic force microscopy, and dynamic light scattering. The polymers self-assembled in solution and complexed plasmid DNA through ionic interactions. The pDNA complexes with 30 lysine residues prepared at a polymer/nucleotide ratio of 10 and with a solution diameter of 380 nm showed the highest efficiency for transfection. The pDNA complexes were also immobilized on silk films and demonstrated direct cell transfection from these surfaces. The results demonstrate the potential of bioengineered silk proteins as a new family of highly tailored gene delivery systems.