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 journalArticle

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

Fingerprint

Staphylococcal Protein A
Protein-Tyrosine Kinases
Fusion reactions
src Homology Domains
Proteins
Periplasm
Mutagenesis
Phosphorylation
Missense Mutation
Site-Directed Mutagenesis
Codon
Purification
Catalyst activity
Bacteria
Phosphotransferases
Adenosine Triphosphate
Experiments
Substrates
Enzymes

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Saya, H., Lee, P. S. Y., Nishi, T., Izawa, I., Nakajima, M., Gallick, G. E., & Levin, V. A. (1993). Bacterial expression of an active tyrosine kinase from a protein A/truncated c-src fusion protein. FEBS Letters, 327(2), 224-230. https://doi.org/10.1016/0014-5793(93)80174-S

Bacterial expression of an active tyrosine kinase from a protein A/truncated c-src fusion protein. / Saya, Hideyuki; Lee, Polly S Y; Nishi, Toru; Izawa, Ichiro; Nakajima, Motowo; Gallick, Gary E.; Levin, Victor A.

In: FEBS Letters, Vol. 327, No. 2, 26.07.1993, p. 224-230.

Research output: Contribution to journalArticle

Saya, H, Lee, PSY, Nishi, T, Izawa, I, Nakajima, M, Gallick, GE & Levin, VA 1993, 'Bacterial expression of an active tyrosine kinase from a protein A/truncated c-src fusion protein', FEBS Letters, vol. 327, no. 2, pp. 224-230. https://doi.org/10.1016/0014-5793(93)80174-S
Saya, Hideyuki ; Lee, Polly S Y ; Nishi, Toru ; Izawa, Ichiro ; Nakajima, Motowo ; Gallick, Gary E. ; Levin, Victor A. / Bacterial expression of an active tyrosine kinase from a protein A/truncated c-src fusion protein. In: FEBS Letters. 1993 ; Vol. 327, No. 2. pp. 224-230.
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