Chaperone-assisted expression and purification of the AML-associated Src-family kinase Fgr in Escherichia coli.

Fgr, a member of the Src family of nonreceptor tyrosine kinases, is expressed in myeloid hematopoietic cells and is a validated drug target in acute myeloid leukemia. Like other Src family kinases, Fgr consists of an N-terminal unique domain, SH3 and SH2 regulatory domains, a catalytic kinase domain, and a C-terminal tail. Notably, Fgr is constitutively active when ectopically expressed in fibroblasts, with kinase activity uncoupled from SH3-SH2 domain regulation, a feature that distinguishes it from other family members. To support structural and biochemical studies, we developed a chaperone-assisted method to express and purify soluble, active Fgr in Escherichia coli. Fgr was coexpressed with C-terminal Src kinase to maintain C-terminal tail phosphorylation and with the protein-tyrosine phosphatase 1B catalytic domain to keep the activation loop dephosphorylated. While this strategy stabilizes soluble Src and Hck in E. coli, it was insufficient for Fgr. To enhance solubility, we added coexpression with the E. coli GroEL-GroES chaperone complex. This significantly improved Fgr solubility, although the kinase initially remained bound to GroEL-GroES. Washing column-bound GroEL-GroES-Fgr with ATP-MgCl partially dissociated the chaperone complex, enabling isolation of pure Fgr through subsequent size-exclusion and ion exchange chromatography. This method reliably produced highly pure, active recombinant Fgr, with consistent yields of 1 mg per 2 l of bacterial culture.