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Öğe Caf1 of Yersinia pestis forms complex highly stable protein polymers and hydrogel scaffolds(Cell Press, 2016) Waller, Helen; Ulusu, Yakup; Lakey, Jeremy H.Caf1 is a polymeric protein from the plague bacterium Y. pestis which is secreted via the chaperone-usher pathway and protects the pathogen from macrophages of the host’s immune system by forming a protective layer around the cell. The 15.5kDa monomer has beta structure and resembles the extracellular matrix protein fibronectin. The polymer is expressed recombinantly in Escherichia coli, secreted by the bacteria forming a flocculent layer above the cell pellet that can be harvested and the polymer extracted in large quantities. An NHS-PEG crosslinker was used to form a stable hydrogel which has potential for development in 3D-tissue culture and regenerative medicine applications due to its low cost, high stability and biodegradable nature. We have selectively reversed the natural non-stick behaviour of the WT polymer by introducing an integrin binding sequence, RGDS, into loop5 and can promote fibroblast adhesion and growth. Here we describe the high stability of the WT protein which confirms its potential as a medical polymer. The polymer is stable on SDS-PAGE gels and this analysis revealed it’s resistance to a range of proteases. Circular dichroism spectroscopy and differential scanning calorimetry revealed it is extremely thermostable from pH2.0 to 11.0 with maximum stability at pH6 of >90 C. It is also stable at high ionic strength and in a range of detergents. Several cell binding motifs have been introduced into the polymer; bone morphogenic protein and collagen motifs (for promoting bone cell adhesion) and laminin motifs (for keratinocyte applications such as wound healing). Degradation sites such as matrix metalloproteinase and thrombin cleavage motifs can be located in regions which promote efficient breakdown of the hydrogel. Co-polymers have been produced by expressing selectively designed monomers off separate plasmids, giving almost limitless possibilities for hydrogel designÖğe Insight into interface engineering at TiO2/dye through molecularly functionalized caf1 biopolymer(Amer Chemical Soc, 2018) Akın, Seçkin; Ulusu, Yakup; Waller, Helen; Lakey, Jeremy H.; Sönmezoğlu, SavaşThe fast charge recombination kinetics and poor sensitizing ability in dye-sensitized solar cells (DSSCs) result in a significant electron loss and performance degradation. However, the retarding of electron recombination and/or increasing light-harvesting efficiency (LHE) via employing an appropriate interface modifier in DSSCs has rarely been investigated. Here, we first report a molecularly engineered Caf1 protein (both in monomeric and polymeric forms) to modify the surface states by effectively shielding the unfavorable reactions and improve the light absorption properties by introducing alternative anchoring facilities. Using the novel Caf1 biopolymer with high thermal stability (even at 90 degrees C), we achieved an unprecedented efficiency of 8.31% under standard illumination test conditions and maintain the output performance even under prolonged irradiation. Time-resolved fluorescence spectroscopy measurement reveals an improved electron transfer rate (k(ET) = 0.26 to 0.98 x 10(8) s(-1)), whereas the V-oc decay rate is lower (70% decay in 90 s) for Caf1-P@TiO2 based cells than that of bare ones (similar to 85% decay in <10 s). We attributed this trend to the presence of chains in the biopolymer structure and the enhanced population of binding facilities with sensitizer molecules, promoting rapid charge transfer into TiO2 and enhanced dye-loading capability. Our results shed light on the interface engineering, and this novel Caf1 biopolymer offers a meaningful transfer of energy to develop efficient electrochemical cells with attractive properties for scale up and practical applications.Öğe Protein engineering of Caf1 from the plague bacterium Yersinia pestis for tissue engineering applications(Wiley, 2016) Peters, Daniel; Ulusu, Yakup; Waller, Helen; Lakey, JeremyInstitute of Cellular and Molecular Bioscience, Medical School, University of Newcastle, UK, 2 Department of Bioengineering, Faculty of Engineering, Karamanoglu-Mehmetbey University, Karaman, Turkey The capsular antigen F1 (Caf1) protein of Y. pestis forms a gel-like, non-stick coat, allowing the bacteria to resist phagocytosis by macrophages. As cells cannot adhere to Caf1, new functions can be engineered in to control cell adhesion, differentiation and proliferation, through the mutation of the protein at key sites. Previously, a mutant Caf1 polymer containing an insertion mutant corresponding to the integrin binding motif (RGD) was produced, which reversed the non-stick phenotype and facilitated the adhesion of cells. Caf1 can also be made to form a hydrogel, highlighting the potential for this protein in tissue engineering applications. Building on this work, we test Caf1’s ability to retain its thermostability under different chemical conditions, and demonstrate its resistance to common proteases. We then show that several regions of the protein can be modified to contain new functional mutations such as growth factor peptides, cell adhesion motifs and protease recognition sites which allow for specific polymer cleavage. Finally, we show the engineered proteins can be combined to form mixed Caf1 polymers with multiple properties, similar to extracellular matrix proteins. The production of defined Caf1 polymers with different functionalities will greatly expand its use as a material in regenerative medicine, for example as a wound care productÖğe Thermal stability and rheological properties of the 'non-stick' Caf1 biomaterial(Iop Publishing Ltd, 2017) Ulusu, Yakup; Dura, Gema; Waller, Helen; Benning, Matthew J.; Fulton, David A.; Lakey, Jeremy H.; Peters, Daniel T.The ability to culture cells in three-dimensions has many applications, from drug discovery to wound healing. 3D cell culture methods often require appropriate scaffolds that mimic the cellular environments of different tissue types. The choice of material from which these scaffolds are made is of paramount importance, as its properties will define the manner in which cells interact with the scaffold. Caf1 is a protein polymer that is secreted from its host organism, Yersinia pestis, to enable escape from phagocytosis. In vitro, cells adhere poorly to the protein unless adhesion motifs are specifically introduced. Caf1 is a good candidate biomaterial due to its definable bioactivity, economical production and its ability to form hydrogels, through the use of cross-linkers. In this study, the thermostability of Caf1 was tested over a range of chemical conditions, and an initial characterisation of its rheological properties conducted in order to assess the suitability of Caf1 as a biomedical material. The results show that Caf1 retains its high thermostability even in harsh conditions such as extremes of pH, high salt concentrations and the presence of detergents. In solution, the concentrated polymer behaves as a complex viscous liquid. Due to these properties, Caf1 polymers are compatible with 3D bioprinting technologies and could be made to form a stimuliresponsive biomaterial that can alter its macrorheological properties in response to external factors. Caf1 biomaterials could therefore prove useful as 3D cell scaffolds for use in cell culture and wound repair.
