title: Expression and characterization of recombinant human retinol-binding protein in Pichia pastoris.
abstract: Plasma retinol-binding protein (RBP4) is the principal carrier of vitamin A in blood. Recent studies have suggested that RBP4 may have also a role in insulin resistance. To date the recombinant protein is usually produced by refolding inclusion bodies in Escherichia coli. Here we report the expression and characterization of recombinant human plasma RBP4 using the Pichia pastoris expression system. Simple and rapid purification allowed us to obtain 5mg/L of purified protein from the fermentation supernatant with no need to perform denaturing and refolding steps. The identity of the protein was verified by ion-trap MS and Western blotting. The functionality of recombinant RBP4, i.e., the binding to its physiologic ligand, retinol, and the interaction with transthyretin (TTR), was tested by fluorimetric and pull-down assays, respectively. The apparent dissociation constant for retinol to the recombinant protein of 2 x 10(-7)M was consistent with published data for native human protein. The recombinant protein interacted specifically with TTR. These results suggest that expression of recombinant human RBP4 in P. pastoris provides an efficient source of fully functional protein in soluble form for biochemical and biophysical studies.
authors.affiliation: The Marie Curie Laboratory for Membrane Proteins, National University of Ireland, Maynooth, County Kildare, Ireland.
authors.forename: Monika
authors.initials: M
authors.lastname: Wysocka-Kapcinska
authors.forename: José Angel
authors.initials: JA
authors.lastname: Campos-Sandoval
authors.forename: Akos
authors.initials: A
authors.lastname: Pal
authors.forename: John B C
authors.initials: JB
authors.lastname: Findlay
mesh_terms: D000595:Amino Acid Sequence; D001671:Biochemistry / Q000379:methods*; D001681:Biological Assay; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D011228:Prealbumin / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D054839:Retinol-Binding Proteins, Plasma / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013997:Time Factors; D014801:Vitamin A / Q000378:metabolism
chemical_list: D011228:Prealbumin; C101326:RBP4 protein, human; D011994:Recombinant Proteins; D054839:Retinol-Binding Proteins, Plasma; D014801:Vitamin A