Journal: Febs Letters
Article Title: The N2-Src neuronal splice variant of C-Src has altered SH3 domain ligand specificity and a higher constitutive activity than N1-Src
Figure Lengend Snippet: Expression and purification of active Src kinases for in vitro kinase assays. (A) Schematic of the GST-PTP1B-His-Δ80Src construct (lacking the first 80 residues of rat full length Src) used to express active Src kinases in E. coli . The 3C protease site to produce His-Src is indicated. (B) Coomassie stained SDS–PAGE gels showing the stages of a representative expression and purification of GST-PTP1B-His-Δ80N1-Src (asterisk; left panel) and its cleavage to yield His-Δ80N1-Src (arrow; middle panel). The right panel shows a comparison of purified His-Δ80C-, N1- and N2-Srcs (arrow). (C) Schematic showing the principles of an in vitro Src kinase assay in which GST-peptide substrates contain a tyrosine residue that can be phosphorylated by the Src kinase domain, and a proline rich sequence inserted at the position of PxxP to assess the effect of SH3 domain binding. The primary sequences of the n-Src loops of the C-, N1- and N2-Src SH3 domains are also depicted. (D) Primary sequences of the GST-peptide substrates used in this study. All substrates contain a phosphorylatable tyrosine residue (*). YP1 and YP2 contain canonical Class I and Class II C-Src SH3 domain binding sequences. YA is a mutant of YP1 in which the prolines have been substituted with alanine (underlined) and YL contains the SH2:kinase linker sequence.
Article Snippet: Following purification with glutathione resin (Genscript), the His-tagged kinases were cleaved from GST-PTP1B by incubation with PreScission 3C protease (GE Healthcare) at 4 °C overnight.
Techniques: Expressing, Purification, In Vitro, Construct, Staining, SDS Page, Kinase Assay, Sequencing, Binding Assay, Mutagenesis