Peptides are a group of molecules consisting of two or more amino acid residues linked by a peptide bond. They are very useful, among other things they play an important role as affinity ligands of immunoglobulins (Ig) and represent a good alternative to natural Ig-binding proteins. Due to their simpler structure compared to proteins, they are more resistant to harsh cleaning conditions of the affinity chromatographic column. In addition, they can be produced cost-efficiently by chemical synthesis. Peptides as affinity ligands bind selectively and relatively strongly to different biological molecules. Specifically, cyclic peptides exhibit high selectivity and binding affinity as they have fewer accessible conformations. Due to their rigidity, they are often better ligands compared to their linear variants (if the binding conformation is similar to the one that the cyclic peptide occupies in the solution, there is only a small decrease in entropy upon target binding, which is thermodynamically favorable). However, not every cyclization necessarily leads to an improved affinity. On the contrary, it may even impair affinity if the conformation becomes unfavorable upon cyclization. Therefore, when designing such peptide variants, it makes sense to gradually examine how certain changes in the peptide structure affect affinity. The bacteriophage display technique is often used to identify high-affinity peptides. It is a robust technique that facilitates the identification of strong and selective peptide ligands for the target macromolecule. In this master's thesis, we investigated the influence of GSYWYNVWF peptide cyclization on its binding affinity. Based on the linear peptide obtained in the previous study, we designed several cyclic variants and assessed their binding affinity for IgG Fc region relative to that of the linear parent peptide. The designed peptides were presented on a bacteriophage and their binding affinity was evaluated by phage ELISA assay. With this approach, we found that the 6,11 Cys peptide with the amino acid sequence GSYWYCVWFGC (where cysteine residues are linked by a disulfide bond) showed a higher affinity for IgG compared to its linear variant. We noticed that changing the cyclization site for one site only greatly affects the conformation of the peptide, which becomes unfavorable and has a large negative effect on binding. Because peptide design is time-consuming and does not necessarily lead to ligands with desired properties, we proposed a partially degenerated oligonucleotide to prepare a phage-displayed combinatorial peptide library to ease the search for cyclic peptides displaying high affinity for IgG. With this approach, we preserve certain amino acids at sites that are important for peptide affinity and all other sites are completely randomized. Specifically, we proposed an oligonucleotide encoding the peptide sequence GXYWYCVWFXC for future testing, with X representing randomized sites where any of the proteinogenic amino acids may occur.
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