Bacterial expression and purification of active hematopoietic cell kinase.

Src family kinases (SFKs) are traditionally purified from eukaryotic expression systems. These expression systems can be costly, yield heterogeneously phosphorylated protein samples and present difficulties when metabolic labeling is required for structural studies. Therefore, many attempts have been made to develop bacterial purification systems for SFKs.

Structural determinants of RhoA binding and nucleotide exchange in leukemia-associated Rho guanine-nucleotide exchange factor.

Rho guanine-nucleotide exchange factors (RhoGEFs) activate Rho GTPases, and thereby regulate cytoskeletal structure, gene transcription, and cell migration. Leukemia-associated RhoGEF (LARG) belongs to a small subfamily of RhoGEFs that are RhoA-selective and directly activated by the Galpha12/13 family of heterotrimeric G proteins. Herein we describe the atomic structures of the catalytic Dbl homology (DH) and pleckstrin homology (PH) domains of LARG alone and in complex with RhoA.

Preliminary structure analysis of the DH/PH domains of leukemia-associated RhoGEF.

Leukemia-associated RhoGEF (LARG) is a multidomain protein that relays signals from Galpha(12/13)-coupled heptahelical receptors to GTPases that regulate the cytoskeleton. To understand the molecular basis of LARG-mediated signal transduction, structural analysis of its DH/PH domains has been initiated. The LARG DH/PH domains have been overexpressed in Escherichia coli as a TEV protease-cleavable fusion protein containing maltose-binding protein and a hexahistidine tag at the N- and C-termini, respectively.