Biochemical Characterization of the Lag-3 Protein, a Negative Regulator of the T-Cell Immune Response

Lissette Sepulveda1, Lilen Uchima1, and Zarixia Zavala2. (1) Industrial Biotechnology Program, Univ of PR Mayaguez campus, Mayaguez, PR 00681, (2) Chemistry, Univ of PR Mayaguez campus, Mayaguez, PR 00681

Abstract

My long-term goal in this laboratory is to characterize the interaction of the regulatory T-cell protein lymphocyte activation gene-3 (LAG-3 or CD223) with its ligand, class II MHC proteins. The LAG-3 protein is expressed in activated natural killer (NK) cells, as well as CD4+ and CD8+ T-cells. This protein plays an important role in negatively regulating T-cell activation and proliferation. LAG-3 is a CD4-homolog that, like CD4, binds to class II MHC proteins. We will perform experiments to determine the affinity and life-time constants between LAG-3 and class II MHC molecules using analytical ultracentrifugation, dynamic light scattering, and surface plasmon resonance. Understanding the biochemical characteristics of the LAG-3: MHC II complex will facilitate the identification of the mechanism by which this protein regulates the T-cell activation. I have successfully made constructs for expression of the protein in E. coli, isolating and purifying it from the inclusion bodies and started optimizing fermentation process to produce the protein in large quantities. I recently started making constructs for expression of the protein in Schreider-2 cells (insect cells).