Optimization of Vascular Endothelial Growth Factor (VEGF) Production at Laboratory Scale from Kluyveromyces lactis Yeast Cells, Development of VEGF Specific ELISA and Biosensor Based Diagnostic Kits
Project no. 21791 · Status: Kapanmış · 2020
Summary
<p>Vascular endothelial growth factor (VEGF) is a biomarker that is pathologically associated with diseases such as cancer, rheumatoid arthritis and Parkinson's, as well as physiological events such as wound healing, bone development, and chemotaxis. Determination of VEGF concentrations is very important in clinical and scientific studies. Within the scope of this project, an antibody-based ELISA and a biosensor were developed for the detection of VEGF165, an important member of the VEGF family. VEGF165 used in this project was purified from <em>Kluyveromyces lactis</em> GG799 yeast cells using recombinant DNA technology. In addition, process optimization was carried out in order to increase the efficiency in VEGF165 production. Since the production of recombinant protein is associated with increased biomass, the components of the medium of <em>K. lactis</em> GG799 yeast cells have been optimized. As a result of the optimization study, it was predicted that optimum biomass (approximately 17 g/L) could be obtained with galactose 53.22 g/L, tryptone 28.16 g/L and yeast extract 14.46 g/L. Sandwich, indirect and direct ELISA systems, which are specific and antibody-based to VEGF165 molecule, have been studied. A direct ELISA-like bioanalytical system prepared for VEGF165 determination has been developed. Lactose p-aminobenzoic acid ligated iron nanoparticle (FeNP) was synthesized as a marker for this analysis. Spectrophotometric measurement method was used as the determination method. Covalent conjugation of the synthesized FeNPs with the VEGF recognition antibody, Altuzan (bevacizumab), was performed using the EDC/NHS chemical. Size measurement, zeta potentials, X-ray Photoelectron spectroscopy (XPS) and Scanning Electron Microscopy (SEM) characterization of the prepared FeNPs and Altuzan/FeNPs were performed. VEGF165 was determined in the linear assay range of 0.5 – 100 ng/mL. In addition, the observable limit of detection (LOD) was calculated as 0.17 ng/mL for VEGF165. The reproducibility of the prepared system and its applicability in the presence of some potential entrepreneurs were tested. Artificial serum was prepared and the detectability of VEGF165 in it was determined.</p>