title: Recombinant Expression, Isotope Labeling and Purification of the Vitamin D Receptor Binding Peptide.
abstract: The vitamin D receptor binding peptide, VDRBP, was overexpressed as a fused form with the ubiquitin molecule in Rosetta(DE3)pLysS, a protein production strain ofEscherichia coliharboring an induction controller plasmid. The fusion protein was bound to the immobilized metal ions, and the denaturation and renaturation of the fusion protein were performed as a part of the purification procedure. After the elution of the fusion protein, the peptide hormone was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The purity of the resulting peptide fragment was checked by MALDI-TOF mass and NMR spectroscopy. The final yields of the target peptide were around 5 and 2 mg per liter of LB and minimal media, respectively. The recombinant expression and purification of this peptide will enable structural and functional studies using multidimensional NMR spectroscopy and X-ray crystallography.
authors.affiliation: Department of Chemistry and Institute for Chemical Biology, Sejong University, Seoul 143-747, Korea ; Department of Biochemistry, University of Wisconsin - Madison, Madison, WI 53706, USA.
authors.forename: Young Kee
authors.initials: YK
authors.lastname: Chae
authors.forename: Kiran
authors.initials: K
authors.lastname: Singarapu
authors.forename: W Milo
authors.initials: WM
authors.lastname: Westler
authors.forename: John L
authors.initials: JL
authors.lastname: Markley
keywords: NMR; Recombinant peptide; Vitamin D receptor