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.
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-His(6)) and its rapid purification by a two-step method. Analyses by size-exclusion column chromatography and analytical ultracentrifugation revealed that the purified CHK-His(6) exists as a monomeric species in solution. Biochemical analyses demonstrated that CHK-His(6) 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-His(6) can be used for future studies to decipher the three-dimensional structure, and regulatory and catalytic properties of CHK.
Chan
Khai-Chew
KC
Department of Biochemistry & Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.
Lio
Daisy Sio-Seng
DS
Dobson
Renwick C J
RC
Jarasrassamee
Boonyarin
B
Hossain
Mohammed I
MI
Roslee
Aainaa K
AK
Ia
Kim K
KK
Perugini
Matthew A
MA
Cheng
Heung-Chin
HC
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D007313:Insecta / Q000166:cytology; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019061:src-Family Kinases
D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C091243:Matk protein, rat; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases