title: Functional expression of N-terminally tagged membrane bound cytochrome P450.
abstract: The introduction of an affinity tag offers an attractive approach to isolation of membrane proteins. The type of affinity tag and its positioning in the protein is determined by the desired subsequent experimental uses of the isolated protein. To minimize the risk of interference, membrane proteins may preferentially be tagged on the side of the membrane that does not harbor the active site. In cytochromes P450, affinity tags have traditionally been introduced at the C-terminal to obtain high expression levels and to avoid translocation of the affinity tag over the membrane bilayer. Using the plant cytochrome P450 CYP79A1 and CYP71E1 as model systems, we demonstrate that a full-length CYP79A1 strepII tagged at the N-terminal expresses well and is able to translocate over the lipid bilayer to produce a functionally active protein that is amenable to affinity purification. The expression level and activity of the N-terminally tagged CYP79A1 protein are very similar to those obtained for the C-terminally tagged version. As an experimental tool, ER luminal tagging is envisioned to offer many advantages in future P450 research work e.g. when catalytic properties of an enzyme or protein-protein interactions are to be investigated.
authors.affiliation: Plant Biochemistry Laboratory, Department of Plant Biology and Biotechnology, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hamann
authors.forename: Tomas
authors.initials: T
authors.lastname: Laursen
authors.forename: Birger Lindberg
authors.initials: BL
authors.lastname: Møller
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D045868:Sorghum / Q000235:genetics; D014443:Tyrosine / Q000378:metabolism
chemical_list: C099517:Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; D009842:Oligopeptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D003577:Cytochrome P-450 Enzyme System; C089817:cytochrome P450TYR