Priprava usmerjene bakteriofagne knjižnice cikličnih peptidov in afinitetni izbor ligandov človeških imunoglobulinov G

Gnidovec, Klemen (author), Bratkovič, Tomaž (mentor), Jenko Bizjan, Barbara (co-mentor)

Monoklonska protitelesa so zaradi velike specifičnosti in afinitete vezave pomemben del terapije in diagnostike številnih bolezni. Proizvajamo jih s tehnologijo rekombinantne DNA v sesalskih celicah, ključna pri njihovi izolaciji iz fermentacijske brozge pa je afinitetna kromatografija. Trenutno najbolj uveljavljen afinitetni ligand za izolacijo monoklonskih protiteles je stafilokokni protein A, vendar ta izkazuje številne pomanjkljivosti, zato se raziskave usmerjajo v iskanje novih molekul z afiniteto do človeških imunoglobulinov. Mednje spadajo tudi kratki peptidni ligandi človeških imunoglobulinov G. V magistrski nalogi smo pripravili usmerjeno knjižnico cikličnih peptidov GXYWYCVWFXC, kjer X predstavlja poljubno proteinogeno aminokislino, predstavljenih na površini bakteriofaga M13 v obliki fuzije s plaščnim proteinom p3. Uporabili smo monovalentno predstavitev na osnovi fagmida pIT2-SL. Vanj smo z metodami molekulskega kloniranja vstavil prepoznavno mesto restriktaze AgeI, s čimer smo omogočili izrez insertov, ki kodirajo peptide, pred sekvenciranjem naslednje generacije. Knjižnične fagmide smo v gostiteljske celice dostavili s pomočjo elektrotransformacije. Pripravili smo optimizirana protokola priprave elektrokompetentnih celic seva E. coli TG1 in elektroporacije, ki nam omogočata rutinsko doseganje učinkovitosti transformacije z izoliranim fagmidom v velikostnem razredu 10^7 transformant na µg fagmidne DNA. Učinkovitost transformacije z ligacijsko zmesjo je nižja, zato bi bila za pripravo knjižnic visoke diverzitete potrebna nadaljnja optimizacija postopkov. S sekvenciranjem naslednje generacije smo določili nukleotidna zaporedja cikličnih peptidov, prisotnih v knjižnici pred in po afinitetnem izboru knjižničnih fagov. Analizirali smo pogostost aminokislinskih ostankov na obeh variabilnih mestih v peptidu ter zastopanost posameznih ciklični peptidov v obogateni knjižnici. Po afinitetnem izboru se je v knjižnici na položaju 10 najbolj povečal delež glutamina, ki ga v E. coli TG1 zapisuje tudi zaključni kodon amber. Poleg tega se je v obogateni knjižnici zmanjšal delež hidrofobnih in aromatskih aminokislinskih ostankov ter povečal delež nekaterih polarnih aminokislinskih ostankov na položaju 2. Med cikličnimi peptidi se je po selekciji najbolj povečal delež peptidov GRYWYCVWFQC, GSYWYCVWFQC, GNYWYCVWFQC, GSYWYCVWFGC in GSYWYCVWFEC, katerih afiniteto do človeških IgG bi bilo smiselno primerjalno ovrednotiti v nadaljnjih raziskavah.

• Language: Slovenian
• Keywords: peptidna knjižnica, predstavitev na bakteriofagu, ciklični peptidi, elektroporacija, imunoglobulini G
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-147027
• Publication date in RUL: 21.06.2023

Secondary language

• Language: English
• Title: Construction of focused cyclic peptide phage library and affinity selection of human immunoglobulin G ligands

Abstract

Due to their high binding affinity and specificity, monoclonal antibodies are widely used to diagnose and treat many diseases. They are produced in mammalian cells with recombinant DNA technology and purified from the cell broth using affinity chromatography. The most commonly used affinity ligand for monoclonal antibody purification is staphylococcal protein A. However, since protein A based chromatography has several drawbacks, research has focused on alternative immunoglobulin affinity ligands, such as short synthetic peptides. In the master’s thesis, we constructed a focused phage display library of cyclic peptides GXYWYCVWFXC, where X represents a randomised site where any of the proteinogenic amino acids can occur. The cyclic peptides were displayed on M13 phage in the form of a fusion with minor capsid protein p3. We used a monovalent phagemid based display. Using molecular cloning techniques, we inserted an AgeI restriction enzyme recognition site into the phagemid vector pIT2-SL, which allowed the inserts encoding the displayed peptides to be excised from the vector before next generation sequencing. Library phagemids were introduced into host cells by electrotransformation. We optimized the protocols for electrocompetent E. coli TG1 preparation and electroporation, which routinely allowed us to achieve a transformation efficiency of more than 10^7 transformants per µg of phagemid DNA. However, the transformation efficiency is much lower for electroporation with a ligation mixture, so further optimization is needed for the production of phage display peptide libraries with high diversity. Using next generation sequencing, we identified cyclic peptide ligands in the library before and after affinity selection. We analysed the abundance of amino acid residues at each of the randomised positions. After affinity selection, glutamine, which in E. coli TG1 is also encoded by the amber stop codon, was present at the highest frequency at position 10. There was also a significant decrease in the frequency of hydrophobic and aromatic amino acid residues and an increase in the frequency of hydrophilic amino acid residues at position 2. The most highly enriched cyclic peptides after selection were GRYWYCVWFQC, GSYWYCVWFQC, GNYWYCVWFQC, GSYWYCVWFGC and GSYWYCVWFEC. The affinities of the highly enriched cyclic peptides for human IgG should be comparatively evaluated in further studies.

• Keywords: peptide library, phage display, cyclic peptides, electroporation, immunoglobulins G