title: Functional expression of the uncomplexed serum retinol-binding protein in Escherichia coli. Ligand binding and reversible unfolding characteristics.
abstract: The serum retinol-binding protein solubilizes the lipophilic vitamin A alcohol and plays an important physiological role in the transport of this compound. The monomeric single-domain protein, the three-dimensional structure of which is known, constitutes a well-characterized member of the lipocalin family of proteins. We report here the functional expression of the apo-protein in Escherichia coli by secretion to the periplasm. The recombinant protein, purified in a single step by metal chelate affinity chromatography, exhibits the same ligand binding characteristics as described for the natural protein. Guanidinium chloride-induced unfolding and refolding experiments suggest that the recombinant retinol-binding protein adopts a stable conformation despite being expressed and purified in the absence of the large hydrophobic ligand. The expression system described here should also be useful for the recombinant production of other lipocalin proteins, thus permitting the elucidation of the structure-function relationships of ligand binding by protein engineering.
authors: Max-Planck-Institut f. Biophysik, Frankfurt, Germany.
authors: H N
authors: HN
authors: Müller
authors: A
authors: A
authors: Skerra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012177:Retinol-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013552:Swine; D014801:Vitamin A / Q000378:metabolism*; D014961:X-Ray Diffraction
chemical_list: D004274:DNA, Recombinant; D011994:Recombinant Proteins; D012177:Retinol-Binding Proteins; D014801:Vitamin A