Development and optimization of the properties of binding proteins recognizing specific cytokines and their receptors

Bc. Johana Roubalíková Erbsová

Master's thesis

Abstract:

Cytokines are signaling protein molecules playing an important role in the immune system. Cytokines modulate signaling pathways or gene expression in target cell by binding to specific cellular receptors. Ability to determine amount of cytokines can contribute to detection of progression of some civilizational diseases such as atherosclerosis, cancer, rheumatoid arthritis, asthma, and others. Antibodies used traditionally for treatment and diagnosis of the mentioned diseases provide several problematic properties. This led to development of protein scaffolds, which can partially replace traditional antibodies because of their improved behavior. Protein scaffolds are small, usually monomeric proteins that show stable structure, better cell membrane penetration ability and better affordability because of Escherichia coli production possibility. The scaffold molecules can be trained to bind to new target molecules, typically other proteins by systematic mutations of amino acid residues by various methods of directed evolution such as phage, ribosome, yeast display, by error-prone PCR, or by in silico design. In this thesis, the second generation of protein scaffold derived binders targeting the clinically important Interleukin-10 (IL-10) was developed using directed evolution methods. For determination of binding affinity of modified scaffolds, IL-10 was produced via Schneider 2 cells insect cell line. The second generation of protein scaffolds was derived from parental variants of F5_WT and G3_WT, which were developed in Biomolecular Recognition Laboratory of the Institute of Biotechnology of the Academy of Sciences of the Czech Republic. Binding affinity of the resulting second-generation protein scaffold F5_A2 to IL-10 was determined by microscale thermophoresis (MST).

Keywords

ELISA, IL-10, Protein scaffold, Directed evolution, Error-prone PCR

Language used: English

Date on which the thesis was submitted / produced: 16. 4. 2023

Thesis defence

• Date of defence: 18. 5. 2023
• Supervisor: RNDr. Pavla Postlerová, Ph.D.
• Reader: Ondřej Vaněk, externi

Czech University of Life Sciences Prague

Faculty of Agrobiology, Food and Natural Resources

Master programme:
Conservation and Use of Natural Resources