A SYNTHETIC BIOLOGY APPROACH TO INVESTIGATE THE INTERACTIONS OF MOUSE CRIPTO

Abstract

The Nodal signaling cascade is a signaling pathway that is conserved in vertebrates and plays a central role in mesendoderm specification. The interactions of the Nodal signaling co-receptor, Cripto, are unclear in the existing literature. We addressed this problem by using a synthetic biology approach that utilizes the site-specifically installed cross-linking amino acid, p-benzoylphenylalanine, to tease out direct interactions in live HEK293 cells. We identified a fundamental flaw in the Cripto literature and determine that using a C-terminally tagged version does not impart the correct membrane localization. We created a library of N-terminally tagged, glycosylated mutants of Cripto containing p-benzoylphenylalanine at specific sites that can pave the way for understanding its direct, physical interactions.