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Rapid and efficient purification of Drosophila homeodomain transcription factors for biophysical characterization.
Orlomoski, Rachel; Bogle, Aaron; Loss, Jeanmarie; Simons, Rylee; Dresch, Jacqueline M; Drewell, Robert A; Spratt, Donald E.
Afiliación
  • Orlomoski R; Gustaf H. Carlson School of Chemistry & Biochemistry, Clark University, 950 Main St, Worcester, MA, 01610, USA; Department of Biology, Clark University, 950 Main St, Worcester, MA, 01610, USA.
  • Bogle A; Gustaf H. Carlson School of Chemistry & Biochemistry, Clark University, 950 Main St, Worcester, MA, 01610, USA; Department of Biology, Clark University, 950 Main St, Worcester, MA, 01610, USA.
  • Loss J; Gustaf H. Carlson School of Chemistry & Biochemistry, Clark University, 950 Main St, Worcester, MA, 01610, USA; Department of Biology, Clark University, 950 Main St, Worcester, MA, 01610, USA.
  • Simons R; Gustaf H. Carlson School of Chemistry & Biochemistry, Clark University, 950 Main St, Worcester, MA, 01610, USA; Department of Biology, Clark University, 950 Main St, Worcester, MA, 01610, USA.
  • Dresch JM; Department of Math & Computer Science, Clark University, 950 Main St, Worcester, MA, 01610, USA.
  • Drewell RA; Department of Biology, Clark University, 950 Main St, Worcester, MA, 01610, USA. Electronic address: rdrewell@clarku.edu.
  • Spratt DE; Gustaf H. Carlson School of Chemistry & Biochemistry, Clark University, 950 Main St, Worcester, MA, 01610, USA. Electronic address: dspratt@clarku.edu.
Protein Expr Purif ; 158: 9-14, 2019 06.
Article en En | MEDLINE | ID: mdl-30738927
Homeodomain transcription factors (HD TFs) are a large class of evolutionarily conserved DNA binding proteins that contain a basic 60-amino acid region required for binding to specific DNA sites. In Drosophila melanogaster, many of these HD TFs are expressed in the early embryo and control transcription of target genes in development through their interaction with cis-regulatory modules. Previous studies where some of the Drosophila HD TFs were purified required the use of strong denaturants (i.e. 6 M urea) and multiple chromatography columns, making the downstream biochemical examination of the isolated protein difficult. To circumvent these obstacles, we have developed a streamlined expression and purification protocol to produce large yields of Drosophila HD TFs. Using the HD TFs FUSHI-TARAZU (FTZ), ANTENNAPEDIA (ANTP), ABDOMINAL-A (ABD-A), ABDOMINAL-B (ABD-B), and ULTRABITHORAX (UBX) as examples, we demonstrate that our 3-day protocol involving the overexpression of His6-SUMO fusion constructs in E. coli followed by a Ni2+-IMAC, SUMO-tag cleavage with the SUMO protease Ulp1, and a heparin column purification produces pure, soluble protein in biological buffers around pH 7 in the absence of denaturants. Electrophoretic mobility shift assays (EMSA) confirm that the purified HD proteins are functional and nuclear magnetic resonance (NMR) spectra confirm that the purified HDs are well-folded. These purified HD TFs can be used in future biophysical experiments to structurally and biochemically characterize how and why these HD TFs bind to different DNA sequences and further probe how nucleotide differences contribute to TF-DNA specificity in the HD family.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Recombinantes de Fusión / Proteínas de Homeodominio / Proteínas de Drosophila Límite: Animals Idioma: En Revista: Protein Expr Purif Asunto de la revista: BIOLOGIA MOLECULAR Año: 2019 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Recombinantes de Fusión / Proteínas de Homeodominio / Proteínas de Drosophila Límite: Animals Idioma: En Revista: Protein Expr Purif Asunto de la revista: BIOLOGIA MOLECULAR Año: 2019 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos