Cloning, Expression, Purification, Crystallization And Preliminary Crystallographic Analysis Of AtMAP4Kα2and AtMO25from Arabidopsis Thaliana

Arabidopsis thaliana MAP4Ks(AtMAP4Ks) belongs to plant serine/threonine protein kinase family. The AtMAP4Ks family includes10members:AtMAP4Kl-10. All of the AtMAP4Ks have a N-terminal catalytic domain and a diversified C-terminal dimerization domain, except for AtMAP4K3whose kinase domain is in the middle of the sequence. They have a conserved motif TFVGTPxWMAPEV. The mammalian homolog of Arabidopsis thaliana MAP4Kalpha2is MST4. Mammalian MST4and AtMAP4Kalpha2share63%sequence identity over the kinase domain. MST4is a member of GCK III subfamily which belongs to the Ste20Ser/Thr protein kinase family, but there is only one member MAP4Kalpha2in Arabidopsis thaliana about GCK III subfamily, so we call the MAP4Kalpha2AtGCK III. Mammalian GCK III is widely involved in biological activity such as cell polarity, cell growth and apoptosis. The mammalian homolog of Arabidopsis thaliana AtMO25is MO25. They share44%sequence identity. So we name it AtMO25. Mammalian MO25is a scaffold protein. It serves as a adaptor protein of GCKIII subfamily, regulating the kinase activity of MAP4Kalpha2.The crystal structure of mammalian MST4and MO25complex is solved through structural biology, and the molecular mechanism of MO25activating MST4is clear. MO25mediates the dimerization of MST4, resulting in the activation of MST4by trans-autophosphorylation.The main results were presented as follows:1. The purification of TEV enzyme. The linking region between AtMAP4Ka2、 AtMO25and his-tag is TEV enzyme sites. TEV enzyme therefore can remove the tag of AtMAP4Ka2、AtMO25.2. Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the kinase domain of AtMAP4Kalpha2from Arabidopsis thiana. We firstly construct three clones, including AtMAP4Ka2full length (1-690aa)、AtMAP4Ka2kinase domain (1-290aa) and AtMAP4Ka2kinase domain with WEF motif (1-338aa). Through protein affinity purification, molecular sieve purification, crystal growth and crystal diffraction, we acquired the diffraction data of the AtMAP4Ka2kinase domain.The crystals diffracted to1.9A resolution and belonged to space group C2221, with unit-cell parameters a.=55.27, b=82.93, c=133.15A.3. Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the kinase domain of AtMO25from Arabidopsis thiana. We construct the full-length of AtMO25, and induced expression at16℃with0.4mM IPTG We purified AtMO25through protein affinity purification and molecular sieve purification. Finally, crystal screening of AtMO25is finished.