Mühendislik Fakültesi, Biyomühendislik Bölümü,Bildiri & Sunum Koleksiyonu
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Güncel Gönderiler
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 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 Development of the Caf1 protein as a multi-functional biomaterial for use in 3D cell scaffolds(Springer, 2017) Peters, D. T.; Dura, G.; Waller, H.; Ulusu, Yakup; Fulton, D. A.; Lakey, J. H.…Öğe Glioblastoma Multiforme Beyin Tümörleri İçin İlaç İletiminde Kullanılabilecek Aptamerlerin Geliştirilmesi(Yayıncı Yok, 2012) Bayraç, Abdullah Tahir; Öktem, Hüseyin Avni; Tan, WeihongBu çalışmada glioblastoma multiforme’yi özel olarak tanıyan aptamer probları negatif seçilim yapılmadan tekrarlı hücre SELEX’i temelli bir prosedürün kullanılmasıyla oluşturulmuştur. Seçilim sonrasında 454 sekanslama teknolojisi SELEX yöntemini izlemek için, biyoenformatik araçlar ise yüksek kapasiteli verilerden yararlanılarak aptamerleri tanımlamak için kullanılmıştır. Özellikle hedef hücreye bağlanabilen ve nanomolar düzeyinde ayrılma sabitine (Kd) sahip bir grup aptamer üretilmiştir. Seçilen aptamerler glioblastoma hücre hattına ait farklı türlere yüksek bağlanma eğilimi gösterirken, diğer kanser hücre hatlarına az ya da hiç bir bağlanma göstermemektedir. Bu çalışmada üretilen aptamerler glioblastoma hücreleri için moleküler işaretleyici keşfi için araç olmanın yanı sıra teşhis için kullanılan prob ve hedeflenmiş ilaç dağıtımı gibi farklı uygulama alanlarında kullanılma potensiyeline sahiptirler.
