International Journal of Biological Macromolecules
Purification and characterisation of the PRH homeodomain: Removal of the N-terminal domain of PRH increases the PRH homeodomain–DNA interaction
Introduction
Homeodomain proteins are key regulators of development in all eukaryotes. The Proline-Rich Homeodomain (PRH) protein (also known as Hex) is a transcription factor [1], [6] that regulates cell differentiation and cell proliferation. PRH functions as a regulator of haematopoiesis [3], [8], [10], [12], [14], [19], [20] and exogenous expression of PRH inhibits the proliferation and transformation of haematopoietic cells of myeloid lineage [12], [19]. However, PRH can also function as an oncoprotein in haematopoietic cells of T-cell lineage [8], [14]. In addition to its role in haematopoiesis, PRH is critical for many processes in embryonic development including embryonic patterning, formation of the head, forebrain, thyroid, liver and heart and in development of the vasculature [2], [4], [7], [11], [17]. The PRH protein consists of an N-terminal proline-rich domain, a central homeodomain that binds to specific DNA sequences and an acidic C-terminal domain. The PRH proline-rich N-terminal domain is required for the inhibition of myeloid cell proliferation and the inhibition of cell transformation [12], [19]. An Engrailed-homology (Eh1) motif within the N-terminal 46 amino acids of PRH recruits members of the Groucho/TLE family of co-repressor proteins [16]. The N-terminal domain also contains amino acid sequences that mediate interactions with PML and translation initiation factor eIF4E. The interaction between PRH and eIF4E inhibits the mRNA transport activity of eIF4E and this leads to regulation of the cell cycle [18], [19]. The PRH homeodomain is a 60 amino acid conserved sequence that binds DNA in a sequence-specific fashion. Homeodomains are composed of three α helices; helices I and II are separated by a loop and helices II and III by a turn. The PRH homeodomain is particularly unusual in that it contains two proline residues at the beginning of the second helix.
Efforts to characterise PRH have been hampered by the proline-rich nature of the protein. However, we have recently described the purification and characterisation of the full-length PRH protein [5]. Here we describe the purification and characterisation of truncated fragments of PRH and we show that removal of the N-terminal domain of PRH increases the DNA binding activity of the PRH homeodomain.
Section snippets
Plasmids used in this study
pTrcAHisPRH is a bacterial expression plasmid that expresses the full-length histidine-tagged avian PRH [5]. pTrcAHisPRH-HDC expresses a histidine-tagged truncated avian PRH construct (amino acids 137–277) that lacks the PRH N-terminus [9]. The plasmid pTrcAHisPRH-HD express a histidine-tagged truncated PRH construct consisting of the avian PRH homeodomain (amino acids 137–205). This plasmid was produced by digesting pTrcAHisPRH-HDC with PstI and EcoRI and inserting an oligonucleotide that
Purification of His-tagged PRH derivatives
We have described previously the purification of His-tagged full-length PRH [5]. The purified His-PRH protein binds to specific DNA sequences poorly. This result is surprising given that a GST fusion protein consisting of the homeodomain and C-terminal domain of PRH fused to GST has been reported to bind DNA readily, although no attempt was made to quantify the binding of this protein to DNA [1], [6]. In order to investigate this apparent disagreement we produced two His-tagged truncated PRH
Discussion
The Proline-Rich Homeodomain protein is essential for normal embryonic development and is also important in the regulation of cell proliferation and gene expression in the adult [9], [15], [19]. The proline-rich nature of this transcription factor has hampered efforts to characterise the protein in detail. Step-gradients are used in the large-scale production of proteins but are used much less commonly in small-scale protein production. Here we have shown that the use of a step-gradient results
Acknowledgements
As is grateful to the Algerian Government for a Ph.D. Scholarship. PSJ is grateful to the MRC for a Career Development Award and The Wellcome Trust for Project Grant funding.
References (20)
- et al.
J. Chromatogr. B
(2003) - et al.
J. Biol. Chem.
(2001) - et al.
Blood
(2003) - et al.
Leuk. Res.
(2000) - et al.
J. Biol. Chem.
(2004) - et al.
Nucl. Acids Res.
(1993) - et al.
Dev. Dyn.
(2000) - et al.
Proc. Natl. Acad. Sci. U.S.A.
(2003) - et al.
Development
(2000) - et al.
Nucl. Acids Res.
(1992)