For enzyme and DSF assays, murine or human Aurora A, MELK (1 to 340), PLK1 (1 to 364), PLK4 (1 to 369), full-length PKA, EPHA3 (kinase domain and juxtamembrane region), ABL (46 to 515), TACC3, and TPX2 (1 to 43) were produced in BL21 (DE3) pLysS E. coli cells (Novagen) with expression induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside for 18 hours at 18°C and purified as N-terminal His6-tag or N-terminal His6-GST tag fusion proteins by affinity chromatography and size exclusion chromatography using a HiLoad 16/600 Superdex 200 column (GE Healthcare) equilibrated in 50 mM tris-HCl (pH 7.4), 100 mM NaCl, and 10% (v/v) glycerol. Where appropriate, recombinant protein was purified in the presence of 1 mM DTT. Ser278Ala, Ser278Cys, Cys290Ala Aurora A, and equivalent Cys-Ala mutants of other kinases were generated using standard mutagenic procedures and purified as described above (66). To generate a phosphorylation-depleted kinase, Aurora A was coexpressed with λPP in BL21 (DE3) pLysS E. coli cells before purification; λPP was subsequently removed by immobilized metal affinity chromatography and size exclusion chromatography.

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