Silk–Silica Composites from Genetically Engineered Chimeric Proteins: Materials Properties Correlate with Silica Condensation Rate and Colloidal Stability of the Proteins in Aqueous Solution

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.

Control of silicification by genetically engineered fusion proteins: Silk–silica binding peptides

“…The silicification reactions were performed according to our prior methods [1,21]. The lyophilized 6mer and chimera proteins were dissolved in double distilled water and then filtered with 5.0 μm syringe filters (Millipore, MA) to remove insoluble precipitates.…”

“…The process of silica precipitation in solution at pH 7.0 was monitored by turbidity at 595 nm [1] with a plate reader Spectra-Max M2 (Molecular Device, CA) from three independent experiments.…”

“…Recently, an artificial silk polymer, 15mer ((SGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGT) 15 , 39 kDa), derived from the consensus repeat of Nephila clavipes dragline silk protein [19,20] was fused with the SiBPs (A1, A3 and R5) and silicification was performed at a series of pHs [1], demonstrating that silica condensation rates and silk–silica composite morphology correlated with colloidal instability of the chimeric proteins which was controlled by pH. In addition, another SiBP Pep1 (KSLSRHDHIHHH), also isolated from phage display, was chemically fused to spider silk polymers, 6mer and 15mer [21], and these polymers showed increased silica condensation compared with the control, silk protein alone.…”

“…Condensed silica nanoscale particles (600–800 nm) were formed in the presence of the recombinant silk and chimeras, which were smaller than those formed by 15mer-SiBP chimeras [1], revealing that the molecular weight of the silk domain correlated to the sizes of the condensed silica particles in the solution system. In addition, the chimeras (6mer-A1/A3/R5) produced smaller condensed silica particles than the control (6mer), revealing that the silica particle size formed in the solution system is controlled by the size of protein assemblies in solution.…”

Biosilica and bioinspired silica studied by solid-state NMR

“…[173] It has been long demonstrated that encapsulation mediated by fusion proteins endowed with silica-forming activity increases entrapment efficiency. [173,174,37,[175][176][177][178][179][180][181][182] We have thus investigated the outcome -in terms of spectral quality, entrapment efficiency, catalytic activity and spectroscopic properties of the matrix -of fusing the R5 peptide to our target enzyme [183].…”