Expression and Purification of a GRAS Domain of Rice GRAS Protein, SLR1, Suitable for Structural Analysis

Abstract

GRAS proteins, which belong to a plant-specific protein family, perform diverse functions in plant growth and development. They are typically composed of a variable N-terminal domain and a highly conserved C-terminal domain, named the GRAS domain. Even though the GRAS domain is considered to play central roles in the biological functions of GRAS proteins, its biophysical investigations have been hampered by the difficulty of preparing samples suitable for such studies. In this report, we describe an over-expression and purification protocol using baculovirus-infected insect cells for the full-length rice DELLA protein, SLR1, which is one of the most extensively studied GRAS proteins and is involved in gibberellin (GA) signaling. Limited proteolysis analyses of the full-length SLR1 indicated that the region including the entire GRAS domain was highly resistant to protease cleavage. Therefore, we constructed an E. coli expression and purification system for the protease resistant GRAS domain and prepared samples for various physicochemical characterization methods, including CD, NMR, and SPR. The results strongly suggested that the GRAS domain adopts a folded structure with abundant secondary structural elements. We also found that a mutant GRAS domain, GRASCA, in which six cysteine residues in the wild-type GRAS are replaced with alanine, exhibits improved properties for physicochemical studies without impairment of its biological functions. Furthermore, we obtained NMR evidence for the direct interactions between the isolated GRAS domains and the GA receptor, GID1. These samples will be suitable for detailed structural and functional studies, which will contribute to further understanding of the plant growth and development mechanisms regulated by the DELLA and GRAS proteins.