Poskus optimizacije peptidnih ligandov regije Fc imunoglobulinov G

Martinšek, Klemen (author), Bratkovič, Tomaž (mentor)

Monoklonska protitelesa omogočajo specifično vezavo antigena, zaradi česar jih uporabljamo v širokem naboru aplikacij. Monoklonska protitelesa praviloma izoliramo in prečistimo z afinitetno kromatografijo na osnovi proteina A, ki ima kljub visoki selektivnosti vezave številne pomanjkljivosti. Zaradi tega razvijajo alternativne ligande, ki so stabilnejši in cenejši. V magistrskem delu smo na osnovi izhodiščnega peptida, pridobljenega v pretekli raziskavi, in rezultatov obogatitve v presejalnem testu zasnovali nove linearne in ciklične peptidne različice peptidnih ligandov, ki bi bili zmožni močnejše vezave na regijo Fc IgG v primerjavi z izhodiščnim peptidom. Zasnovane peptide smo predstavili na bakteriofagu v monovaletnem formatu in primerjalno ovrednotili njihove vezavne afinitete s testi ELISA. Rezultati s cikličnimi različicami peptida so bili nekonsistentni, zato smo ciklične različice opustili. Linarne različice peptidov smo podvrgli nizu testov ELISA, s katerimi smo postopno izločali zasnovane peptide. Za najuspešnejšega smo določili peptid GSYWYNVWF. Izbiro peptida smo potrdili z enosmerno analizo variance ANOVA, ki je pokazala na statistično značilne razlike v afiniteti vezave med izbranim peptidom in ostalimi različicami. Izvedli smo tudi bioinformacijsko analizo zasnovanih peptidov z izračunom teoretične izoelektrične točke, indeksa GRAVY in indeksa nestabilnosti, s katerimi smo podali možno razlago vzrokov za vezavne razlike med peptidi.

• Language: Slovenian
• Keywords: Imunoglobulini, bakteriofagni prikaz, peptidi, ligandi
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [K. Martinšek]
• Year: 2021
• PID: 20.500.12556/RUL-125893
• UDC: 606.68:602.3:578.347:602.44:543.544.17:577.112(043.2)
• COBISS.SI-ID: 61878275
• Publication date in RUL: 09.04.2021

MARTINŠEK, Klemen, 2021, Poskus optimizacije peptidnih ligandov regije Fc imunoglobulinov G [online]. Master’s thesis. Ljubljana : K. Martinšek. [Accessed 1 June 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=125893

Secondary language

• Language: English
• Title: Optimisation of peptide ligands of immunoglobulin G Fc region

Abstract

Monoclonal antibodies allow specific antigen binding, which is why they are used in a wide range of applications. Monoclonal antibodies are currently isolated and purified mainly by using protein A-based affinity chromatography, which, despite its high binding selectivity, has a number of disadvantages. As a result, efforts are being made to develop alternative ligands that are more cost-efficient and stable. In this master's thesis, we designed new linear and cyclic peptide variants based on the starting peptide obtained in previous research and the results of enrichment during library screening. The goal was to identify peptide ligands with improved binding affinity for the Fc IgG region. The designed peptides were presented on a bacteriophage and their binding affinities were comparatively evaluated with ELISA assays. The results of cyclic variants were contradictory, so cyclic variants were not pursued further. The linear variants were subjected to a series of ELISA assays during which the peptides were gradually excluded. The GSYWYNVWF peptide was determined to have optimal binding affinity. Peptide selection was confirmed by one-way ANOVA assay, which showed statistically significant differences in binding affinity between the selected peptide and other variants. We performed a bioinformatical analysis (i.e., calculation of theoretical isoelectric point, GRAVY and instability indices), which gave a possible explanation of the reasons for the binding differences among the peptides.

• Keywords: Immunoglobulins, phage display, peptides, ligands