Purification and characterisation of the PRH homeodomain: Removal of the N-terminal domain of PRH increases the PRH homeodomain-DNA interaction.
The Proline-Rich Homeodomain (PRH) protein is a regulator of transcription and translation and plays a key role in the control of cell proliferation and cell differentiation. PRH contains an N-terminal proline-rich domain that can repress transcription when expressed as a fusion protein with an unrelated DNA binding domain, a central homeodomain that binds to specific DNA sequences and an acidic C-terminal domain of no known function. In order to investigate the structure and functions of PRH we have purified the full-length protein and truncated proteins corresponding to different domains of PRH fused to histidine tags. Here we compare the effects of elution conditions and column volume on protein purification and we investigate the DNA binding activity of these proteins. We show that the PRH homeodomain co-purifies with nucleic acids even after nuclease treatment and that a high salt-wash is required to remove bound nucleic acids. In contrast with the full-length PRH protein, the PRH homeodomain binds to DNA with high affinity. We show that a truncated protein comprising the homeodomain and C-terminal domain also binds to DNA with high affinity and we conclude that the N-terminal domain of PRH inhibits the homeodomain-DNA interaction.
Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
Abdenour
A
Soufi
Kevin
K
Gaston
Padma-Sheela
PS
Jayaraman
D000595:Amino Acid Sequence / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
C078492:HHEX protein, human; D018398:Homeodomain Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA