Histamine Bağlanan Peptitlerin Faj Gösterim Yöntemi İle Belirlenmesi

Abstract

Histamine, one of the most frequently found biogenic amines in foods, threatens public health due to its high toxicity. Therefore, rapid, and sensitive quantification of histamine in food samples has vital importance in terms of human health and food safety. Bioassays and biosensors, in which histamine specific enzymes or antibodies are used as biological recognition agents, allow rapid, sensitive, and easy detection of histamine. However, the difficulties in the production of antibodies against low molecular weight targets such as histamine, reduces their applicability. The laborious and costly production of biological recognition molecules and easy loss of their activity due to changes in environmental conditions, bring up the development of artificial recognition agents and their use in bioassays. Therefore, there is an urgent need for the development of histamine binding artificial recognition agents to be used in bioassays. Phage display technology has been used frequently in the selection and development of novel recognition agents that show affinity to various target molecules. Today, phage display technology is used to identify v peptide ligands that bind selectively to many low molecular weight organic molecules with high affinity. In this study, it was aimed to identify peptides that bind specifically to a food-borne chemical intoxication agent, histamine, using the phage display method. For this purpose, the 12mer phage display peptide library was added to BSA-histamine immobilized microtiter well and incubated. Then the non-binding phages were discarded by washing and the bound phages were eluted from the wells. The same panning cycle was repeated for three times after amplification of the bound phages. In the last panning cycle, histidine binding phages were eliminated by adding the histidine amino acid, which has a similar structure to histamine for enhancing the selectivity of the histamine binding phages. Thus, 41 phage clones thought to have different peptide sequences that bind to histamine, were isolated. The affinity of phage clones to histamine was examined by the phage-ELISA method and DNAs belonging to 7 phage clones showing high affinity were isolated and peptide sequences were determined. Following the sequence analysis, 4 peptides (HBF5, HBF10, HBF14, and HBF26) with the highest affinity and different sequences were synthesized and the thermodynamic properties of their binding properties were determined by using isothermal titration calorimetry. It was observed that HBF10 peptides with SGFRDGIEDFLW peptide sequence and HBF26 with the sequence of IPLENQHKIYST showed high affinity for histamine, and both peptides did not show an affinity for histidine amino acid, which is similar in structure to histamine. The secondary structures of the HBF26 peptide were examined using circular dichroism spectroscopy. The circular dichroism spectrum could not be obtained due to the low solubility of the HBF10 peptide in water. At the end of the study, it is thought that the HBF10 and HBF26 peptides determined by using the phage display method have the potential to be used in various bioassay and biosensor systems for the determination of histamine.