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

FEBS Letters

Lee, Polly S.Y.²  Izawa, Ichiro²  Gallick, Gary E.¹  Nakajima, Motowo¹  Levin, Victor A.²  Nishi, Toru²  Saya, Hideyuki² 

[1] Department of Tumor Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA;Department of Neuro-Oncology The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA

关键词: c-src;    Protein A;    Protein tyrosine kinase;    Escherichia coli;    PTK;    protein tyrosine kinase;    SH2;    src homology region 2;    PCR;    polymerase chain reaction;    EDTA;    ethylenediaminetetraacetic acid;    SDS;    sodium dodecylsulfate;    PAGE;    polyacrylamide gel electrophoresis;    Glu;    glutamic acid;    Tyr;    tyrosine;    PBS;    phosphate buffered saline;    ECL;    enhanced chemiluminescence;    Ile;    isoleucine;    Thr;    threonine;    ATP;    adenosine triphosphate;    Lys;    lysine;    His;    histidine;    Arg;    arginine;   

DOI  :  10.1016/0014-5793(93)80174-S

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown