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

Hideyuki Saya, Polly S.Y. Lee, Toru Nishi, Ichiro Izawa, Motowo Nakajima, Gary E. Gallick, Victor A. Levin

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)224-230
Number of pages7
JournalFEBS Letters
Volume327
Issue number2
DOIs
Publication statusPublished - 1993 Jul 26
Externally publishedYes

Keywords

  • Escherichia coli
  • Protein A
  • Protein tyrosine kinase
  • c-src

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

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