Yakup Ulusu

Thermal stability and rheological properties of the ?non-stick? Caf1 biomaterial

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 stimuli-responsive 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.

Bacillus subtilis’e Ait Ksilanaz (xyn-akky1)’in Escherichia coli’de Klonlanması, Ekspresyonu ve Karakterizasyonu

Bu calismada, Bacillus subtilis akky1 straini Turkiye’nin Ordu Ili’nin Akkus ilcesindeki kayin ormanindan alinan topraktan izole edilmistir. akky1 straini 16S rRNA analizi ile tanimlanmistir. 16S rRNA dizisi Bacillus subtilis strain B7 (KC310823.1) ile %100’luk bir benzerlik gostermistir. GenBank’da verilen Bacillus subtilis ksilanazina ait gen dizisi esas alinarak tasarlanan primerler kullanilarak genomic DNA’dan 642 bp’lik bir DNA fragmenti elde edilmistir. Ksilanazi kodlayan gen, pET28b (+) ekspresyon vektorune klonlanmis ve Escherichia coli BL21 (DE3)’de ekpresse edilmistir. Rekombinant olarak uretilen protein nikel afinite kromatografisi ile saflastirilmis ve ksilanaz aktivitesi belirlenmistir. Saflastirilan ksilanazin molekuler agirligi SDS-PAGE ile 26.0 kDa olarak belirlenmistir. Rekombinant enzimin optimum pH’si 6.0 ve sicakligi 60°C, Km degeri ise 3.33 mg/ml olarak tespit edilmistir.

Expression, purification, and characterization of bovine chymosin enzyme using an inducible pTOLT system

ABSTRACT In recent years, various studies in the field of industrial enzymes of biotechnology have gained importance due to increasing development in enzyme technology. The different areas where enzymes are used and their economic value of biotechnological products further increases their importance. There are hundreds of different types of cheese but each is made by coagulating milk using rennet to give curds. Today, researchers have begun to develop alternative systems in the cheese industry related to milk-clotting enzymes. In this study, the nucleic acid sequence encoding the optimized chymosin enzyme was used and cloned by Not I and Mlu I restriction enzymes into pTOLT vector system. Then using this construct, the enzyme as a fusion with Tol-A-III protein was produced in Escherichia coli BL21 (DE3) cells. After disrupting the E. coli cell and separating from the constituents by high speed centrifugation, the enzyme was purified by affinity chromatography and fractions were analyzed by SDS–PAGE. Purified enzyme has shown its activity. Optimum temperature and pH of CHY-Tol-A-III protein were 40°C and 6.5, respectively.

Protein engineering of Caf1 from the plague bacterium Yersinia pestis for tissue engineering applications

Grouping by Topics Pages PA – Amyloid and Aggregation 9 PB – Bioinformatics 19