Investigation of mutations (L41F, F17M, N57E, Y99F_Y134W) effects on the TolAIII-UnaG fluorescence protein's unconjugated bilirubin (UC-BR) binding ability and thermal stability properties.

The UnaG protein is a ligand (unconjugated bilirubin) dependent fluorescence protein isolated from Unagi freshwater eel larvae and expressed as fusion in heterologous expression systems. Bilirubin is a tetrapyrrole molecule mainly produced from heme catabolism by the destruction of erythrocytes in the body. Bilirubin can cause kernicterus, a serious condition associated with permanent neurological damage in neonates with the passage of brain tissue. Different methods have been developed for plasma bilirubin analysis and quantification. The use of UnaG fluorescence protein triggered by bilirubin has become a new approach in bilirubin studies. In this study, we aimed to investigate the biophysical characterization of ligand interactions with the proteins obtained as a result of mutations (UnaGY99F_Y134W, UnaGN57E, UnaGL41F, and UnaGF17M) on the amino acid sequence of TolAIII-UnaG protein. After the purity levels of the expressed proteins have been analyzed by SDS-PAGE, secondary structures and thermal melting temperatures of the proteins have been examined by circular dichroism spectroscopy. Then determination of excitation and emission points by fluorescence spectroscopy, titration studies have been performed with bilirubin, and dissociation constant was calculated. According to the biophysical characterization studies, UnaGL41F has the highest affinity and stability among the mutants.

Recovery and Reusability of ApoUnaG Fluorescence Protein from the Unconjugated Bilirubin Complex Structure.

This study is the first report on the separation and reusability of ApoUnaG protein, indicating excellent fluorescence response with high affinity and specificity toward unconjugated bilirubin (UC-BR) molecules, from the UnaG-UC-BR complex structure. The fluorescence properties of the UnaG-UC-BR complex (holo-UnaG) are studied by addition of different metal ions to perform possible interactions with holo-UnaG through absorbance and emission spectra. After addition of metal ions, some changes with respect to the type of metal ions are observed in fluorescence intensity of the holo-UnaG. When compared to metal ions, an excellent quenching response is sighted in the presence of Cu2+ ions by binding with UC-BR in the UnaG-UC-BR complex structure. Obtained non-fluorescence holo-UnaG-Cu2+ complex mixture is passed through Ni-NTA agarose to remove the ingredients such as Cu2+, UC-BR and Cu2+-UC-BR coordination complex from holo-UnaG. From the obtained experiments, it is concluded that Cu2+ ion can be used as an agent for the recovery of ApoUnaG protein via binding with UC-BR molecules. Graphical Abstract Recovery and Reusability of ApoUnaG Fluorescence Protein from the Unconjugated Bilirubin Complex Structure.

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

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.

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.