Murat Topuzogullari

Conjugation, characterization and toxicity of lipophosphoglycan-polyacrylic acid conjugate for vaccination against leishmaniasis

Research on the conjugates of synthetic polyelectrolytes with antigenic molecules, such as proteins, peptides, or carbohydrates, is an attractive area due to their highly immunogenic character in comparison to classical adjuvants. For example, polyacrylic acid (PAA) is a weak polyelectrolyte and has been used in several biomedical applications such as immunological studies, drug delivery, and enzyme immobilization. However, to our knowledge, there are no studies that document immune-stimulant properties of PAA in Leishmania infection. Therefore, we aimed to develop a potential vaccine candidate against leishmaniasis by covalently conjugating PAA with an immunologically vital molecule of lipophosphoglycan (LPG) found in Leishmania parasites. In the study, LPG and PAA were conjugated by a multi-step procedure, and final products were analyzed with GPC and MALDI-TOF MS techniques. In cytotoxicity experiments, LPG-PAA conjugates did not indicate toxic effects on L929 and J774 murine macrophage cells. We assume that LPG-PAA conjugate can be a potential vaccine candidate, and will be immunologically characterized in further studies to prove its potential.

The Fluorescence Study of Interaction Between Bovine Serum Albumin and Polyacrylic Acid

The complex formation of Bovine Serum Albumin (BSA) with anionic polyelectrolyte (polyacrylic acid, PAA) in aqueous solution was studied by a fluorescence technique, pH titration and HPLC analysis. The character of the interactions and solubility of the polycomplex particles depends on the BSA/PAA ratios and the pH of solution. The interaction at pH > pI (isoelectric point of BSA) (pH 6.0–7.0) is negligible weak and at pH 5.0 results with the formation of stable water-soluble polycomplexes at a wide range of protein/polymer ratios. The fluorescence intensity of BSA sharply decreased when an different amount of PAA was added and its maximum wavelength shifts towards the blue region. The protein molecules in the structure of soluble polycomplex particles are densely covered by the shelf of a polymer coil and practically “fenced off” from the water environment. This effect was reinforced by the increase of protein components. Existence of soluble and insoluble PAA-BSA complexes have been observed at pH < pI (pH 4.0–4.3). These soluble complexes characterized by the structure of particles in which protein molecules are densely covered by the shelf of a polymer coil. By the increase in the protein concentration, these complexes aggregate to an interpolymer species.

Thermo‐Responsive Complexes of c‐Myc Antisense Oligonucleotide with Block Copolymer of Poly(OEGMA) and Quaternized Poly(4‐Vinylpyridine)

Solution behavior of thermo-responsive polymers and their complexes with biological macromolecules may be affected by environmental conditions, such as the concentration of macromolecular components, pH, ion concentration, etc. Therefore, a thermo-responsive polymer and its complexes should be characterized in detail to observe their responses against possible environments under physiological conditions before biological applications. To briefly indicate this important issue, thermo-responsive block copolymer of quaternized poly(4-vinylpyridine) and poly(oligoethyleneglycol methyl ether methacrylate) as a potential nonviral vector has been synthesized. Polyelectrolyte complexes of this copolymer with the antisense oligonucleotide of c-Myc oncogene are also thermo-responsive but, have lower LCST (lower critical solution temperature) values compared to individual copolymer. LCST values of complexes decrease with molar ratio of macromolecular components and presence of salt. Dilution of solutions also affects solution behavior of complexes and causes a significant decrease in size and an increase in LCST, which indicates possible effects of severe dilutions in the blood stream.

Comprehensive structural analysis of the open and closed conformations of Theileria annulata enolase by molecular modelling and docking

Theileria annulata is an apicomplexan parasite which is responsible for tropical theileriosis in cattle. Due to resistance of T. annulata against commonly used antitheilerial drug, new drug candidates should be identified urgently. Enolase might be a druggable protein candidate which has an important role in glycolysis, and could also be related to several cellular functions as a moonlight protein. In this study; we have described three-dimensional models of open and closed conformations of T. annulata enolase by homology modeling method for the first time with the comprehensive domain, active site and docking analyses. Our results show that the enolase has similar folding patterns within enolase superfamily with conserved catalytic loops and active site residues. We have described specific insertions, possible plasminogen binding sites, electrostatic potential surfaces and positively charged pockets as druggable regions in T. annulata enolase.

Cloning, expression and characterization of the gene encoding the enolase from Fusobacterium nucleatum

The gene encoding enolase from Fusobacterium nucleatum (FnENO) was cloned and analyzed for the first time. The gene comprises of 1302 nucleotide base pairs and encodes 433 amino acids. The gene sequence alignment demonstrated the presence of several distinct insertions and deletions, compared with the human enzyme. The gene for recombinant FnENO was inserted into the pLATE 31 vector system and expressed in E. coli BL21(DE3) cells as a soluble protein. The protein was purified by affinity chromatography using a Ni-NTA agarose matrix and shown on SDS-PAGE to be a 46 kDa protein. The molecular weight of the octameric form of the purified recombinant protein was determined as being 375 kDa by size exclusion chromatography. Optimal enzyme activity was observed at pH 8.5 and the enzyme remained stable at a range of different temperatures from 30 to 60°C. Using 2-phosphoglyceric acid as substrate for the purified enzyme, KM, kcat and kcat/KM were determined as 0.48 mM, 20.4 s–1 and 4.22 × 104 M–1s–1, respectively. Potential drug binding sites of FnENO were detected using homology modeling. These data could facilitate the design of new inhibitors of F. nucleatum which has already been shown to be resistant to several known antibiotics.

Thermal Destabilization of Rhizomucor miehei Rennet with Aldehyde Dextran Sulfate: Purification, Bioconjugation and Milk-Clotting Activities

High thermal stability of Rhizomucor miehei Rennet, which is a thermostable enzyme used in cheese production, causes undesired cases at elevated temperatures. This study aims to decrease the thermal stability of the R. miehei Rennet at high temperatures. To achieve this goal, bioconjugates of R. miehei Rennet with aldehyde derivative of dextran sulfate were synthesized in different molar ratios. Physico-chemical properties of bioconjugates were characterized with particle size analyzer and gel permeation chromatography (GPC) techniques. The enzyme and biopolymer were conjugated with medium efficiency. Milk-clotting activities of bioconjugates decreased drastically at high temperatures in all molar ratios, which reveals that covalent bioconjugation of the enzyme with aldehyde derivative of dextran sulfate caused a decrease in thermal resistance of this enzyme.