title: Purification and characterization of a DNA-binding recombinant PREP1:PBX1 complex.
abstract: Human PREP1 and PBX1 are homeodomain transcriptional factors, whose biochemical and structural characterization has not yet been fully described. Expression of full-length recombinant PREP1 (47.6 kDa) and PBX1 (46.6 kDa) in E. coli is difficult because of poor yield, high instability and insufficient purity, in particular for structural studies. We cloned the cDNA of both proteins into a dicistronic vector containing an N-terminal glutathione S-transferase (GST) tag and co-expressed and co-purified a stable PBX1:PREP1 complex. For structural studies, we produced two C-terminally truncated complexes that retain their ability to bind DNA and are more stable than the full-length proteins through various purification steps. Here we report the production of large amounts of soluble and pure recombinant human PBX1:PREP1 complex in an active form capable of binding DNA.
authors: FIRC (Foundation for Italian Cancer Research) Institute of Molecular Oncology (IFOM), via Adamello 16, 20139, Milan, Italy.
authors: Lisa
authors: L
authors: Mathiasen
authors: FIRC (Foundation for Italian Cancer Research) Institute of Molecular Oncology (IFOM), via Adamello 16, 20139, Milan, Italy.
authors: Chiara
authors: C
authors: Bruckmann
authors: Crystallography Unit, Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, Milan, 20139, Italy.
authors: Sebastiano
authors: S
authors: Pasqualato
authors: FIRC (Foundation for Italian Cancer Research) Institute of Molecular Oncology (IFOM), via Adamello 16, 20139, Milan, Italy.
authors: Francesco
authors: F
authors: Blasi
mesh_terms: D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005822:Genetic Vectors / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D046912:Multiprotein Complexes; C106682:PKNOX1 protein, human; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C083400:PBX1 protein, human; D005982:Glutathione Transferase