Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/71454
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Type: Journal article
Title: Bacterial expression and purification of active hematopoietic cell kinase
Author: Kristelly, R.
Qiu, W.
Gunn, N.
Scanlon, D.
Mulhern, T.
Citation: Protein Expression and Purification, 2011; 78(1):14-21
Publisher: Academic Press Inc Elsevier Science
Issue Date: 2011
ISSN: 1046-5928
1096-0279
Statement of
Responsibility: 
Romana Kristelly, Theresa Wenli Qiu, Natalie J. Gunn, Denis B. Scanlon, Terrence D. Mulhern
Abstract: 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 plays an important role in phagocyte function as well as the etiology of chronic myeloid leukemia as Hck is an interaction partner of Bcr-Abl. Structural studies of Hck are essential to fully understand the signaling processes involved in host defense and leukemogenesis. Successful bacterial expression of Hck was possible by a dual strategy: (1) co-expression with YopH phosphatase in order to control host toxicity, and (2) expression in a bacterial strain that is RNase E deficient, which dramatically increased overall expression levels. The expressed Hck construct is unphosphorylated and appears to be in an open conformation. Bacterially expressed Hck is capable of autophosphorylation, phosphorylates substrate at rates comparable to insect cell expressed Hck, and can be inhibited by staurosporine and Csk.
Keywords: Src-family kinase; Hematopoietic cell kinase; Bacterial expression; Kinase assay; Enzyme inhibition
Rights: Copyright © 2011 Elsevier Inc. All rights reserved.
RMID: 0020110795
DOI: 10.1016/j.pep.2011.03.001
Grant ID: http://purl.org/au-research/grants/nhmrc/509115
Appears in Collections:IPAS publications

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