Data for the co-expression and purification of human recombinant CaMKK2 in complex with calmodulin in Escherichia coli

Abstract

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 'apo', CaMKK2 (165-501) in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, "Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2" [1].

Keywords: AMPK, adenosine monophosphate-regulated kinase; CaM, calmodulin; CaMK, calcium/calmodulin-dependent kinase; CaMKK2; CaMKK2, calcium/calmodulin-dependent kinase kinase 2; CaMKK2:CaM, CaMKK2-CaM complex; Calmodulin; E. coli, Escherichia coli; Fermentation; IPTG, β-D-1-thiogalactopyranoside; LB, Luria broth; LDS-PAGE, lithium dodecyl sulphate–polyacrylamide gel electrophoresis; LIC, ligation-independent cloning; OD, optical density; PMSF, phenylmethylsulfonyl fluoride; SEC, size-exclusion chromatography; TEV, tobacco etch virus; aa, amino acid.

Fig. 1

Workflow diagram. CaMKK2-pET30-Ek/LIC and CaM-pET8a were co-transformed into E. coli Rosetta™ II (DE3) and overexpression performed as indicated. LDS-PAGE shows samples taken before induction and 18 hours after induction; M=molecular weight marker. Bands corresponding to overexpressed CaMKK2 and CaM proteins are marked. The His₆-tagged full-length CaMKK2 protein (541 aa) with its kinase domain (KD) (aa 165-446), arginine-proline-rich site (RP) (aa 204-226), autoinhibitory domain (AID) (aa 472-477), CaM binding domain (aa 475-500). The binding of CaM to CaMKK2 is Ca²⁺-dependent and consequently, CaCl₂ was included throughout expression and purification. Protein purification was achieved as outlined. Affinity chromatography was followed by TEV-cleavage and dialysis. After a second affinity chromatography step the CaMKK2:CaM complex was subjected to size exclusion chromatography.

Fig. 2

Purification of the CaMKK2:CaM protein complex. M=molecular weight marker. Bands corresponding to overexpressed CaMKK2 and CaM proteins are marked with an asterisk.

Fig. 3

His-TEV test digest. M=molecular weight marker.

Fig. 4

Purification of apo-CaMKK2. M=molecular weight marker. Bands corresponding to overexpressed CaMKK2 protein are marked with an asterisk.