Analysis of c - Src kinase mutants for understanding conformational plasticity

............................................................................................................................ 3 Introduction ...................................................................................................................... 4 Protein tyrosine kinases ............................................................................................... 4 Discovery and oncogenicity of c-Src ............................................................................ 6 Structural domains of c-Src .......................................................................................... 6 Conformational plasticity ............................................................................................. 8 Aim of the work........................................................................................................... 10 Materials and Methods ................................................................................................... 11 Plasmid ....................................................................................................................... 11 Design of mutations .................................................................................................... 11 Site directed mutagenesis ........................................................................................... 13 Transformation of Escherichia coli competent cells .................................................. 14 DNA sequencing and analysis .................................................................................... 14 Co-transformation in Escherichia coli BL21DE3 strain............................................ 15 Protein expression ...................................................................................................... 15 Protein purification .................................................................................................... 15 Verification of absence of post-translational modifications ...................................... 17 Western blot ................................................................................................................ 17 Kinase assay and inhibition of protein activity by imatinib ....................................... 18 Results ............................................................................................................................ 20 Discussion....................................................................................................................... 29 Concluding remarks........................................................................................................ 33 References ...................................................................................................................... 34