Expression and purification of a GRAS domain of SLR1, the rice DELLA protein.
GRAS proteins belong to a plant specific protein family that participates in diverse and important functions in growth and development. GRAS proteins are typically composed of a variable N-terminal domain and highly conserved C-terminal GRAS domain. Despite the importance of the GRAS domain, little biochemical or structural analyses have been reported, mainly due to difficulties with purification of sufficient quality and quantity of protein. This study is focused on one of the most extensively studied GRAS proteins, the rice DELLA protein (SLR1), which is known to be involved in gibberellin (GA) signaling. Using a baculovirus-insect cell expression system we have achieved overproduction and purification of full-length SLR1. Limited proteolysis of the full-length SLR1 indicated that a region including the entire GRAS domain (SLR1(206-625)) is protease resistant. Based on those results, we have constructed an expression and purification system of the GRAS domain (SLR1(206-625)) in Escherichia coli. Several physicochemical assays have indicated that the folded structure of the GRAS domain is rich in secondary structural elements and that alanine substitutions for six cysteine residues improves protein folding without impairing function. Furthermore, by NMR spectroscopy we have observed direct interaction between the purified GRAS domain and the GA receptor GID1. Taken together, our purified preparation of the GRAS domain of SLR1 is suitable for further structural and functional studies that will contribute to precise understanding of the plant regulation mechanism through DELLA and GRAS proteins.
Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Tomomi
T
Sato
Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
Yohei
Y
Miyanoiri
Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
Mitsuhiro
M
Takeda
Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Youichi
Y
Naoe
Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan.
Rie
R
Mitani
Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan.
Ko
K
Hirano
Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan.
Sayaka
S
Takehara
Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan; Center for Priority Areas, Graduate School of Science and Technology, Tokyo Metropolitan University, 1-1, Minami-ohsawa, Hachioji 192-0397, Japan.
Masatsune
M
Kainosho
Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan.
Makoto
M
Matsuoka
Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan.
Miyako
M
Ueguchi-Tanaka
Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address: katohiro@pharm.kyoto-u.ac.jp.
Hiroaki
H
Kato
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D012275:Oryza / Q000235:genetics*; D010446:Peptide Fragments; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014357:Trypsin
D010446:Peptide Fragments; D010940:Plant Proteins; D011994:Recombinant Proteins; D014357:Trypsin
DELLA protein; GA signaling; GRAS domain; Limited proteolysis; NMR; SLR1