RESUMEN
The tendency of bacteria to assemble at oil-water interfaces can be utilized to create microbial recognition sites on the surface of polymer beads. In this work, two different groups of bacteria were first treated with acryloyl-functionalized chitosan and then used to stabilize an oil-in-water emulsion composed of cross-linking monomers that were dispersed in aqueous buffer. Polymerization of the oil phase followed by removal of the bacterial template resulted in well-defined polymer beads bearing bacterial imprints. Chemical passivation of chitosan and cell displacement assays indicate that the bacterial recognition on the polymer beads was dependent on the nature of the pre-polymer and the target bacteria. The functional materials for microbial recognition show great potential for constructing cell-cell communication networks, biosensors, and new platforms for testing antibiotic drugs.
Asunto(s)
Bacterias/citología , Quitosano/análogos & derivados , Emulsiones/química , Impresión Molecular/métodos , Aceites/química , Polímeros/química , Polimerizacion , Propiedades de Superficie , Agua/químicaRESUMEN
Spider silk has been studied extensively for its attractive mechanical properties and potential applications in medicine and industry. The production of spider silk, however, has been lagging behind for lack of suitable systems. Our approach focuses on solving the production of spider silk by designing, expressing, purifying and characterizing the silk from cylindriform glands. We show that the cylindriform silk protein, in contrast to the commonly used dragline silk protein, is fully folded and stable in solution. With the help of GFP as a fusion tag we enhanced the expression of the silk protein in Escherichia coli and could optimize the downstream processing. Secondary structures analysis by circular dichroism and FTIR shows that the GFP-silk fusion protein is predominantly α-helical, and that pH can trigger a α- to ß-transition resulting in aggregation. Structural analysis by small angle X-ray scattering suggests that the GFP-Silk exists in the form of a hexamer in solution.
Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Multimerización de Proteína , Seda/química , Animales , Dicroismo Circular , Dispersión Dinámica de Luz , Electroforesis en Gel de Poliacrilamida , Fluorescencia , Concentración de Iones de Hidrógeno , Modelos Moleculares , Replegamiento Proteico , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/aislamiento & purificación , Dispersión del Ángulo Pequeño , Solubilidad , Espectroscopía Infrarroja por Transformada de Fourier , Arañas , Difracción de Rayos XRESUMEN
Amelogenin is an extracellular protein first identified as a matrix component important for formation of dental enamel during tooth development. Lately, amelogenin has also been found to have positive effects on clinical important areas, such as treatment of periodontal defects, wound healing, and bone regeneration. Here we present a simple method for purification of recombinant human amelogenin expressed in Escherichia coli, based on the solubility properties of amelogenin. The method combines cell lysis with recovery/purification of the protein and generates a >95% pure amelogenin in one step using intact harvested cells as starting material. By using amelogenin as a fusion partner we could further demonstrate that the same method also be can explored to purify other target proteins/peptides in an effective manner. For instance, a fusion between the clinically used protein PTH (parathyroid hormone) and amelogenin was successfully expressed and purified, and the amelogenin part could be removed from PTH by using a site-specific protease.