[53] Production of p60c-src by baculovirus expression and immunoaffinity purification
Publisher Summary
This chapter describes an effective method for the large-scale production of p60c-src, a 60-kDa protein-tyrosine kinase associated with the inner face of cellular membranes. It emphasizes the general considerations that should be used for applying this technique to the study of any protein kinase or other enzyme. For expression of p60c-src , the baculovirus system, employs the Autographa californica nuclear polyhedrosis virus (AcMNPV), a baculovirus that can be used to infect Spodoptera frugiperda (sf9) insect cells in culture. Late in the lytic virus life cycle, the viral polyhedrin protein is expressed at extremely high levels (over 50% of cell protein), resulting in the formation of crystalline viral occlusions in the infected cell. The purification approach employs columns containing MAb 327, a monoclonal antibody that binds with moderately high affinity and high specificity to an amino-terminal epitope on p60c-src from several species. For largescale purification (up to l0 mg of p60c-src), a column is developed by binding 80 mg of protein A-purified MAb 327 to 10 ml protein A-Sepharose. The antibody is then covalently coupled to the protein A with dimethyl pimelimidate. This method provides maximum binding capacity, since the antibody on the column is theoretically oriented with all of its antigen binding sites accessible to the antigen.
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A rapid ATP affinity-based purification for the human non-receptor tyrosine kinase c-Src
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PTPH1 Is a Predominant Protein-tyrosine Phosphatase Capable of Interacting with and Dephosphorylating the T Cell Receptor ζ Subunit
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