Bacterial expression and purification of active hematopoietic cell kinase
Romana Kristelly, Theresa Wenli Qiu, Natalie J Gunn, Denis B Scanlon, Terrence D Mulhern
Protein Expression and Purification | ACADEMIC PRESS INC ELSEVIER SCIENCE | Published : 2011
Src family kinases (SFKs) are traditionally purified from eukaryotic expression systems. These expression systems can be costly, yield heterogeneously phosphorylated protein samples and present difficulties when metabolic labeling is required for structural studies. Therefore, many attempts have been made to develop bacterial purification systems for SFKs. So far, high-yield bacterial expression systems have only been achieved for SFK kinase domains or for inactive mutants of constructs containing the regulatory SH3 and SH2 domains, but not for their active forms. Herein described is a bacterial expression system for the wild type, active SFK Hck containing SH3, SH2 and kinase domains. Hck p..View full abstract
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Awarded by National Health and Medical Research Council
We are indebted to Heung-Chin Cheng and John Kuriyan for the provision of the pcDNA3 Hck and YopH plasmids, respectively. We thank Daisy Sio Seng Lio for technical assistance and Nicholas Williamson for assistance with the analysis of the mass spectrometry data. This work was supported by a University of Melbourne ANSTO Collaborative Grant (RK and TDM) and by the National Health and Medical Research Council Project Grant 509115 (TDM).