Generation and Characterization of Peptide Fusion Proteins

Author(s)

Brianna L. Probasco, Rowan University

Date Approved

2013

Document Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

Department

Cell Biology

College

Graduate School of Biomedical Sciences

Funder

Humigen LLC; Medical Diagnostic Laboratories

First Advisor

Grant Gallagher, PhD

Committee Member 1

Raymond Yu, PhD

Committee Member 2

Venkataswar Venkataraman, PhD

Committee Member 3

Kathleen Iacono, PhD

Subject(s)

Th17 Cells, Autoimmune Diseases, Interleukin-23, Peptides, Gene Expression Regulation

Disciplines

Cell Biology | Genetic Processes | Immune System Diseases | Laboratory and Basic Science Research | Medical Cell Biology | Medicine and Health Sciences | Molecular Biology | Therapeutics

Abstract

Pathogenic Th17 cells drive progression of many autoimmune diseases. Th17 cells develop from naïve T cells in the immune system after antigen-driven stimulation in a specific cytokine environment. Normally, T cells act to fight off infection, but when not properly controlled, they can cause disease. The cytokine interleukin-23 (IL-23) plays an essential role in the expansion of pathogenic Th17 cells. IL-23 is a heterodimeric protein, composed of a p19 alpha chain and a p40 beta chain. The p40 is also part of IL-12 and binds to the IL-12 receptor beta 1 (IL-12Rβ1) subunit. Thus, it follows that the IL-23 receptor is comprised of the IL-12Rβ1 and IL-23 receptor alpha (IL23Rα) subunits. New research in therapeutics for autoimmune disease is attempting to inhibit IL-23 from binding to its receptor. Our laboratory previously screened a peptide library through display technologies for peptides that bound to the IL-23Rα. Peptides #7 and #2 were identified as having the strongest inhibitory activity on the IL-23 signaling pathway. However, peptides are small, leading them to be cleared from a biological system in a manner of minutes; therefore, the present project created fusion proteins of these peptides with mouse IgG2a-Fc. They were then expressed in mammalian cells, secreted and purified from medium. Peptide #2-Fc and its unmodified counterpart, Peptide #2, were tested and compared using cell-free and cell-based systems to ensure minimal loss of inhibitory activity upon fusion. This work generated a Peptide #2-Fc fusion protein with the capability to bind the IL23Rα chain and thus block IL-23 binding and inhibit subsequent signaling, as well an increased serum half-life over the original Peptide #2.

Recommended Citation

Probasco, Brianna L., "Generation and Characterization of Peptide Fusion Proteins" (2013). Graduate School of Biomedical Sciences Theses and Dissertations. 7.
https://rdw.rowan.edu/gsbs_etd/7