Screening and Developing Adhiron Molecules That Bind BTB Protein Binding Domains of ZBTB Transcription Factors
6th INTERNATIONAL MOLECULAR IMMUNOLOGY & IMMUNOGENETICS CONGRESS (MIMIC-VI), İstanbul, Turkey, 28 - 30 April 2025, (Unpublished)
- Publication Type: Conference Paper / Unpublished
- City: İstanbul
- Country: Turkey
- Acibadem Mehmet Ali Aydinlar University Affiliated: Yes
Abstract
ZBTB proteins are a family of transcription factors that control critical biological processes such as cell differentiation, proliferation, immunoglobulin class switching and the epigenetic regulation of lymphocytes. This highly conserved protein family is associated with multiple malignancies and a rare disease known as Immunodeficiency, Centromeric Instability and Facial Anomalies (ICF) syndrome. This syndrome results from the deficiency of the ZBTB24 protein. Deciphering the functions and molecular interactions of these ZBTB proteins can shed light on developmental processes in the human body and pave the way of novel diagnosis and therapy modalities. We aim to develop affinity reagents, alternative to antibodies. By using molecular engineering and library selection, we develop molecules with high binding specificity that can be used for imaging, detection or molecular inhibition. We created a yeast surface display library of an artificially engineered protein called an adhiron. Adhirons are derived from pytocystatins and their small size, temperature stability and the possibility of bacterial expression make them attractive reagents. We have screened this yeast adhiron display library to find novel binders against the BTB domain of the ZBTB24 protein, using three rounds of magnetic selection followed by three rounds of cell sorting (FACS). We have successfully selected yeast cells expressing ZBTB24 binding adhirons effective at target protein concentrations of 100nM. Sequence analysis of adhiron plasmid DNA from sorted yeast populations revealed that we have identified novel adhirons binding to the ZBTB24 protein. These clones are in the process of being expressed in E.coli and will be characterized in detail. Our future studies include developing these adhirons as detection reagents for the screening of the presence of ZBTB24 protein in ICF Syndrome patients.