Production and Characterization of Odorant Binding Proteins

• Autor(a) principal: Calvário, Joana Ramos dos Santos
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/133791
• topic: Odorant Binding Proteins; insect OBPs; expression; purification; gas sensing; VOCs; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Odorant Binding Proteins (OBPs) are small soluble proteins responsible for the binding, solubilization and transportation of Volatile Organic Compounds (VOCs) to olfactory receptors in vertebrates and insects. These proteins have been gaining attraction in artificial olfaction fields due to their natural selectivity toward VOCs. Furthermore, OBPs are reported to be structurally stable, resistant to thermal and proteolytic degradation, and easily produced in E. coli. Therefore, it is possible to implement OBPs in biosensors in order to develop a sensor array with improved selectivity by mimicking the biological olfactory system. The main objective of this work is the expression, purification and characterization of insect OBPs, for further application in an electronic nose biosensor. The design of a vector construct with OBP genes under an IPTG inducible promoter allowed the proteins’ recombinant expression in E. coli. A first expression test revealed the presence of OBPs as inclusions bodies (IBs). Further expression strategies were tested to improve protein solubility during expression, however proved to be ineffective. Thus, CPI (Contact Pathway Inhibitor) from Phlebotomus duboscqi and OBP56a from Phormia regina were selected, expressed as IBs, solubilized and purified. Different purification protocols were assessed. Immobilized Metal Affinity Chromatography (IMAC) in denaturing conditions demonstrated to be the most successful approach, with purities of 86% (CPI) and 92% (OBP56a), and recoveries of 71% (CPI) and 50% (OBP56a). Western blot confirmed the presence of monomeric and dimeric forms of the purified OBPs. The secondary structure of the alpha helix rich insect OBPs was assessed by Circular Dichroism. We consider that further optimizations are still required, mainly in dialysis and a second purification step. Lastly, preliminary results were obtained in gas sensing experiments, in which OBP56a functionalized sensor has shown to be selective toward acetic acid. Overall, this work explored different techniques regarding expression and purification of insect OBPs in inclusion body form. With further optimization, these proteins can be employed in VOC detecting biomaterials, for applications such as the early detection of viral and bacterial infections, cancers and inflammatory diseases.