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
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)
- et al.
Identification and characterization of a novel tyrosine kinase from megakaryocytes
J. Biol. Chem.
(1994) - et al.
CSK: a protein-tyrosine kinase involved in regulation of Src family kinases
J. Biol. Chem.
(1991) - et al.
Endogenous and synthetic inhibitors of the Src-family protein tyrosine kinases
Biochim. Biophys. Acta
(2005) - et al.
Expression, purification, and biochemical characterization of Chk, a soluble protein tyrosine kinase
Protein Expr. Purif.
(2003) - et al.
A novel non-catalytic mechanism employed by the C-terminal Src-homologous kinase to inhibit Src-family kinase activity
J. Biol. Chem.
(2004) - et al.
A protein tyrosine kinase involved in regulation of pp60c-Src function
J. Biol. Chem.
(1989) - et al.
Structural basis for the recognition of c-Src by its inactivator Csk
Cell
(2008) - et al.
C-terminal Src kinase-homologous kinase (CHK), a unique inhibitor inactivating multiple active conformations of Src family tyrosine kinases
J. Biol. Chem.
(2006) Src family kinases: regulation of their activities, levels and identification of new pathways
Biochim. Biophys. Acta
(2008)- et al.
RhoB and actin polymerization coordinate Src activation with endosome-mediated delivery to the membrane
Dev. Cell
(2004)
Modulation of the catalytic activity of the Src family tyrosine kinase Hck by autophosphorylation at a novel site in the unique domain
J. Biol. Chem.
Self-association of human apolipoprotein E3 and E4 in the presence and absence of phospholipid
J. Biol. Chem.
Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling
Biophys. J.
Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems
Biophys. J.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
Quantification of Coomassie Blue stained proteins in polyacrylamide gels based on analyses of eluted dye
Anal. Biochem.
Assay of cyclic AMP-dependent protein kinases
Methods Enzymol.
Isolation of phosphorylated peptides and proteins on ion exchange papers
Anal. Biochem.
Functions of the activation loop in Csk protein–tyrosine kinase
J. Biol. Chem.
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