Abstract:

Mutation（F80A）on CDK2 kinase allows the selective use of an ATP analogue N⁶-(2-phenylethyl)-ATP (PE-ATP), which exhibits high affinity for the altered CDK2. Using modified CDK2 and its ATP analogue enables the detection of novel CDK2 substrates in cells. Modified CDK2 （F80A）myc was cloned into pCMV vector, and transfected into HeLa cells by polyfect transfection reagent. The cells expressing mutant CDK2 were selected by the ability of resistance to G418. The stably transfected cell line was identified by immunoblotting. Four out of the six mutants showed CDK2(F80A)myc expression, and two of them got a higher expression level. The higher CDK2(F80A)myc expressing cell lines can be used for screening CDK2 substrates in cells.

Key words: stable cell line； CDK2； mutation； apoptosis