Development of the procedures for high-yield expression and rapid purification of active recombinant Csk-homologous kinase (CHK): Comparison of the catalytic activities of CHK and CSK

https://doi.org/10.1016/j.pep.2010.07.007Get rights and content

Abstract

Csk-homologous kinase (CHK) is an important endogenous inhibitor constraining the oncogenic actions of Src-family kinases (SFKs) in cells. It suppresses SFK activity by specifically phosphorylating the conserved regulatory tyrosine near the C-terminus of SFKs. In addition to phosphorylation, CHK employs a novel non-catalytic inhibitory mechanism to suppress SFK activity. This mechanism involves direct binding of CHK to the active forms of SFKs to form stable protein complexes. Since aberrant activation of SFKs contributes to cancer formation and progression, small-molecule inhibitors mimicking the non-catalytic inhibitory mechanism of CHK are potential anti-cancer therapeutics. Elucidation of the catalytic and regulatory properties and the structural basis of the CHK non-catalytic inhibitory mechanism would facilitate the development of these small-molecule inhibitors. To this end, we developed procedures for higher level expression in insect cells of active recombinant CHK with a hexa-histidine tag attached to its C-terminus (referred to as CHK-His6) and its rapid purification by a two-step method. Analyses by size-exclusion column chromatography and analytical ultracentrifugation revealed that the purified CHK-His6 exists as a monomeric species in solution. Biochemical analyses demonstrated that CHK-His6 exhibits efficiencies comparable to those of CSK in phosphorylating artificial protein and peptide substrates as well as an intact SFK protein. Our results indicate that the recombinant CHK-His6 can be used for future studies to decipher the three-dimensional structure, and regulatory and catalytic properties of CHK.

Section snippets

Materials

The “kinase-dead” [K275M]Lyn mutant was generated and purified by procedures as described in our previous report [20]. Recombinant CSK was expressed in Sf9 cells infected with the CSK-baculovirus (from Dr. David Morgan) and purified procedures described previously [24]. The rabbit polyclonal anti-pTyr-507(Lyn) phosphospecific antibody directing against the consensus C-terminal tail phosphorylation site of Lyn was from Cell Signaling, Inc. Poly(Glu4/Tyr1), a random copolymer of glutamate and

Expression and purification of CHK-His6

The infected Sf9 cells were extracted twice by homogenization; the soluble (referred to as “the Extract” in this manuscript) and insoluble (pellet) fractions were separated by centrifugation. Western blot analysis with anti-CHK antibody revealed that the soluble fractions contained most if not all the recombinant CHK-His6 (data not shown). As shown in Fig. 1, CHK-His6, of an apparent molecular mass of ∼53 kDa, is one of the several major proteins in the Extract. In contrast, CHK-His6 is the most

Discussion

In this manuscript, we described the development of the improved procedures for high-yield expression in insect cells and rapid purification of recombinant CHK-His6. The purification procedures involve the use of a DEAE anion exchange column step to bind most of the contaminating proteins in the crude cell lysate, followed by purification of CHK-His6 in the DEAE column flow-through and wash fractions by Ni2+–NTA affinity column chromatography. We routinely obtained 5–7 mg of CHK-His6 of over 95%

Conclusions

In summary, the procedures described in this manuscript allow the production of milligram quantity of highly purified, active and monomeric recombinant CHK-His6 for future structural and biophysical studies to elucidate the regulatory and catalytic properties of CHK. Results of these future studies will provide a much clearer picture of the molecular basis of CHK catalysis and its ability of specific inhibition of Src-family kinases. Since CHK is a specific inhibitor of SFKs in mammalian cells,

Acknowledgements

The works described in this manuscript were supported by project grants of the National Health and Medical Research Council of Australia and the Cancer Council Victoria. We wish to thank Dr. David Morgan of University of California, San Francisco for his generous gift of the recombinant CSK-baculovirus. We wish to thank Dr. Nick Williamson of the mass spectrometry and peptide synthesis core facility of the Bio21 Institute for help in molecular weight determination of the recombinant proteins by

References (39)

View full text