Expression and purification of the human homeodomain oncoprotein HOX11.
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 (HOX11 Delta H3) which lacked the DNA-recognition helix (helix 3) of the homeodomain. Through a single purification procedure using glutathione-Sepharose, 2mg 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 degrees 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-HOX11 Delta H3, to DNA containing consensus HOX11 recognition sites.
Heidari
Mansour
M
Division of Veterinary and Biomedical Sciences, Western Australian Biomedical Research Institute, Murdoch University, Murdoch, WA 6150, Australia.
Rice
Kim L
KL
Kees
Ursula R
UR
Greene
Wayne K
WK
D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005982:Glutathione Transferase; D018398:Homeodomain Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D015513:Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C071250:TLX1 protein, human; D005982:Glutathione Transferase