title: Production of a soluble and functional recombinant apolipoproteinD in the Pichia pastoris expression system.
abstract: ApolipoproteinD (ApoD) is a human glycoprotein from the lipocalin family. ApoD contains a conserved central motif of an 8-stranded antiparallel β-sheet, which forms a beta-barrel that can be used for transport and storage of diverse hydrophobic ligands. Due to hydrophobic nature of ApoD, it has been difficult to generate a recombinant version of this protein. In the present work, we aimed at the production of ApoD in the robust Pichia pastoris expression system. To this end, the ApoD gene sequence was synthesized and subcloned for expression in the yeast host cells. Following integration of the ApoD gene into the yeast genomic region using homologous recombination, the ApoD recombinant protein was induced using methanol, reaching its maximum induction at 96 h. Having purified the ApoD recombinant protein by affinity chromatography, we measured the dissociation constant (KD) using its natural ligands: progesterone and arachidonic acid. Our results provide a viable solution to the production of recombinant ApoD protein in lieu of previous obstacles in generating soluble and functional ApoD protein.
authors.affiliation: Metabolic Disorder Research Center, Golestan University of Medical Science, Gorgan, Iran; Department of Chemistry, Sharif University of Technology, Tehran, Iran.
authors.forename: Shiva
authors.initials: S
authors.lastname: Armanmehr
authors.affiliation: Department of Chemistry, Sharif University of Technology, Tehran, Iran. Electronic address: Kalhor@sharif.edu.
authors.forename: Hamid Reza
authors.initials: HR
authors.lastname: Kalhor
authors.affiliation: Department of Microbiology, School of Medicine, Golestan University of Medical Science, Gorgan, Iran.
authors.forename: Alijan
authors.initials: A
authors.lastname: Tabarraei
mesh_terms: D053399:Apolipoproteins D / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D053399:Apolipoproteins D; D008024:Ligands; D011994:Recombinant Proteins
keywords: Apolipoprotein D; Ligand binding; Pichia pastoris; Recombinant protein