Expression and purification of JAZ3 protein and a transcriptional factor MYC2 from Arabidopsis thaliana

Abstract

Jasmonate is a plant hormone that regulates development, growth and wound response. There are many jasmonate derivatives in nature. Particularly, jasmonyl isoleucine serves as a bioactive molecule in the jasmonate signaling pathway. In fact, the jasmonate signaling is regulated by interactions among four different proteins, including MYC2, JAZ, NINJA, and TOPLESS. Among them, MYC2 and JAZ have critical role in the jasmonate signaling pathway. JAZ binds to MYC2 and represses the jasmonate signaling. Especially, intermolecular interaction of JAZ and MYC2 is attributable to JID motif of MYC2 and Jas motif of JAZ. Nonetheless these interactions play a crucial role in the jasmonate signaling pathway, molecular details of JAZ and MYC2 remain unknown, mainly due to the insoluble properties of each JAZ and MYC2 protein in vitro. In this thesis, I report various approaches that could yield a protein crystal of Jas and JID motif in JAZ and MYC2, respectively. Eight fusion proteins were produced. In those fused proteins, two different constructs for Jas motif and four for JID region are linked using Tobacco Etch Virus (TEV) protease cleavage sequence. Various chromatographic analysis indicated that JAZ forms a complex with MYC2 by cleaving the TEV protease cleavage site. Although crystallization of the complex has not been achieved at this moment, this study provides valuable information for producing the JAZ-MYC2 complex in vitro.