Arvo building, auditorium F115, address: Arvo Ylpön katu 34.
Doctoral defence of M.Sc. Soili Lehtonen
The field of science of the dissertation is Medical technology and biotechnology.
The opponent is docent Kari Airenne (University of Eastern Finland). Professor emeritus Markku Kulomaa acts as the custos.
The language of the dissertation defence is Finnish.
Development of alternative binding proteins
Antibodies, the specific binding proteins produced by our immune system for virtually any foreign target molecule, have been used as valuable laboratory reagents in the life sciences and as therapeutic drugs in medicine for almost a century. However, in recent years these antibody-based affinity reagents have been challenged by novel types of binding proteins developed through directed evolution, offering preferable properties in certain applications. The aim of this thesis was to develop avidin scaffold-based novel binding proteins for small molecule targets, which are challenging to develop high-affinity antibodies for. Avidin is an exceptionally stable protein from chicken egg-white, known for binding exceptionally tightly its native ligand, biotin (vitamin B7). This is the srongest non-covalent interaction known to nature between a protein and its target molecule. The structure-function of avidin is well known, and avidin is known to tolerate genetic modifications well, especially when located in the loop regions. Above all, the binding properties of lipocalins belonging to the same “calycin” superfamily as avidin, have already been successfully modified.
For to develop novel binding proteins, the same methods were used as for developing antibodies in laboratory. Proteins are constructed of amino acids that are attached to one another by peptide bond and arranged based on the encoding DNA sequence. Targeting the loop region amino acid residues involved in biotin-binding for random mutagenesis, libraries consisting of millions of different avidin variants were constructed. In order to select avidin variants based on their binding properties, phage display was used: The avidin variants were displayed on the surface of the bacterial infecting viruses, (bacterio)phages, enabling the selection of variants showing binding towards desired target molecule and their easy amplification in bacterial cells. Testosterone was chosen as the first target molecule, resulting in the selection of avidin variant with micromolar affinity. By genetically modifying the variant further, the remaining biotin-binding affinity was signicantly reduced. Later three different avidin libraries were pooled together and used for the selection of binders towards several diagnostically relevant small molecules in parallel. These developed small-molecule binding avidin variants are called as antidins. The obtained results showed that avidin scaffold has potential to be used as an alternative scaffold for binding several different small molecules.
As a result of this PhD project, new methods for library construction were also established.
The dissertation is published in the publication series of Acta Universitatis Tamperensis; 2305, Tampere University Press, Tampere 2017. The dissertation is also published in the e-series Acta Electronica Universitatis Tamperensis; 1809, Tampere University Press 2017.