Bacterial expression of an active tyrosine kinase from a protein A/truncated c‐src fusion protein

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Abstract
The carboxy‐terminal half of the c‐src protein fused to the protein A moiety was expressed in bacteria. The protein A/truncated c‐src fusion protein, which does not have SH2 and SH3 domains, is found in the periplasmic space allowing for a simple one‐step purification and demonstrated high efficiency in autophosphorylation and exogeneous substrate phosphorylation. The missense mutation at codon 294 (Ile → Thr), which is located in the ATP‐binding domain of the c‐src, resulted in dramatic reduction of tyrosine kinase activity of the fusion protein. Using the fusion protein. we also revealed that staurosporin, a well‐known kinase inhibitor, directly affects autophosphorylation of the C‐terminal half of the c‐src protein. This truncated c‐src expression system provides a good source of enzyme for diverse experiments and is an ideal model for understanding the implication of structural alterations in the catalytic activity of the c‐src kinase by site‐directed mutagenesis experiments.