Expression, purification and characterization of the plant Snf1-related protein kinase 1 from Escherichia coli.

ABSTRACT

The SnRK1 (SNF1 related protein kinase 1), a plant homologue of SNF1 (Sucrose non-fermenting 1)/AMPK (AMP-activated protein kinase), is an important metabolic sensor involving in catabolic and anabolic processes. SnRK1 is essential for plant metabolism regulation and response to environmental stresses. The plant SnRK1 consists of one catalytic (α1/α2 subunit) and two regulatory subunits (ß1/ß2/ß3 and γ/ßγ subunits), and functions as a heterotrimeric complex. Here we took advantage of a tricistronic expression vector and successfully purified the holoenzyme containing three subunits of SnRK1 from the E.coli. Using advantages of the E.coli system, we would be able to purify SnRK1 complex with high yield and high purity. Moreover, the complex is stable with high homogeneity. Using the purified complex, we confirmed that the ßγ rather than the γ subunit of the plant SnRK1 acts as the canonical regulatory subunit. Besides, some basic characters of the SnRK1 holoenzyme was studied. Together, our results provide a convenient way for purify the plant SnRK1 complexes, and this would be helpful for follow-up study on SnRK1's structure and mechanism.