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Expression and purification of the human homeodomain oncoprotein HOX11

Heidari, M., Rice, K.L., Kees, U.R. and Greene, W.K. (2002) Expression and purification of the human homeodomain oncoprotein HOX11. Protein Expression and Purification, 25 (2). pp. 313-318.

Link to Published Version: http://dx.doi.org/10.1016/S1046-5928(02)00014-1
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Abstract

HOX11 is a transcription factor belonging to the homeodomain family that is essential for spleen development during embryogenesis. It is also tumorigenic, being associated with T-cell acute lymphoblastic leukemia in children. In order to understand the functional role of HOX11 in both normal development and malignancy, protein-DNA and protein-protein interaction studies involving this factor are required. Such investigations would be facilitated by the availability of significant amounts of purified HOX11 protein. However, expression of full-length HOX11 in bacteria has been reported to be problematic owing to fusion protein instability. Here, we report the purification of human HOX11 expressed in Escherichia coli as a soluble and functional glutathione S-transferase (GST) fusion protein. In addition, a mutant version of HOX11 was produced (HOX11ΔH3) which lacked the DNA-recognition helix (helix 3) of the homeodomain. Through a single purification procedure using glutathione-Sepharose, 2 mg of the recombinant proteins were obtained per liter of bacterial culture. Notably, recombinant GST-HOX11 fusion proteins had a markedly higher stability when purified at low temperature (4°C). Purification to near-homogeneity was achieved as judged by SDS-PAGE and the purified proteins were recognized by anti-HOX11 antibodies. The biological activity of the recombinant protein was verified by the specific binding of GST-HOX11, but not GST-HOX11ΔH3, to DNA containing consensus HOX11 recognition sites.

Publication Type: Journal Article
Murdoch Affiliation: School of Veterinary and Biomedical Sciences
Publisher: Academic Press
Copyright: © 2002 Elsevier Science (USA).
URI: http://researchrepository.murdoch.edu.au/id/eprint/6856
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