Background

The GRAS proteins are a large family that plays vital roles in plant development and signaling transduction. Findings indicate that some family members such as DELLAs function as a repressor of GA responsive plant growth and are key regulatory targets in the GA signaling pathway (Murase et al., 2008), NSP1 and NSP2 play important roles in regulating nodulation development and signaling (Kaló et al., 2005), the proteins SCR and SHR together play an important role in the control of radial patterning for both the root and shoot (Helariutta et al., 2000), AtLAS is a key regulator in the developmental processes of the axillary meristem (Greb et al., 2003), HAM functions in shoot meristem maintenance (Stuurman et al., 2002).

Based on sequence analyses, GRAS proteins include a variable N-terminal domain and a widely and highly conserved C-terminal domain known as the GRAS domain. The N-terminal domains constitute a plant-specific unfoldome and may act as molecular bait by initiating the key molecular recognition events (Uversky et al., 2010). And the C-terminal GRAS domain is highly conserved in the whole GRAS family, suggesting that these proteins share a similar function and/or a common mode-of-action (Sun et al., 2012).

Though many members of GRAS proteins have been studied, the functional mechanism of GRAS proteins is still unclear. Structural descriptions of GRAS proteins may deeply clarify the functional mechanism of this family. However as yet little structural analyses have been reported, mainly due to the difficulties in obtaining sufficient quality and quantity of GRAS proteins. Soto et al. (2014) reported the expression and purification of the GRAS domain of rice SLR1. They constructed a GST-SLR1 fusion protein and expressed it in E. coli. But the expression levels were low (0.5 mg [TB medium] or 0.2 mg [M9 medium] of purified protein from 1 L flask culture). With some modifications, they obtained 1-3 mg of stable isotope labeled purified protein at 87% purity from 1 L of fermenter culture. However, the expression levels and purity of SLR1 are both not enough for crystallization. Moreover, reducing the protein synthesis rate by low culture temperature and speed, co-expressing with chaperonins, expressing as a fusion protein with soluble tags such as glutathione S-transferase, thioredoxin and maltose-binding protein or mutating some hydrophobic or disulfide forming amino acids are usually used to improve the expression and solubility of target proteins. While using more purification methods and steps will help to improve the purity of target proteins. In order to get a high purity and quantity of GRAS protein, we established a protocol for easy expression and purification of the GRAS domain of Os-SCL7 from rice.