title: Improved method for assembly of hemeprotein neuronal NO-synthase heterodimers.
abstract: The assembly of mutated and wild type monomers into functional heterodimeric hemeproteins has provided important mechanistic insights. As in the case of NO synthase (NOS), the existing methods to make such heterodimeric NOSs are inefficient and labor intensive with typical yields of about 5%. We have found that expression of neuronal NOS heterodimers in insect cells, where we take advantage of an exogenous heme-triggered chaperone-assisted assembly process, provides an approximately 43% yield in heterodimeric NOS. In contrast, in Escherichia coli little heterodimerization occurred. Thus, insect cells are preferred and may represent a valuable method for assembly of other dimeric hemeproteins.
authors.affiliation: Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, MI, 48109-0632, United States.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Morishima
authors.affiliation: Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, MI, 48109-0632, United States.
authors.forename: Haoming
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, MI, 48109-0632, United States.
authors.forename: Miranda
authors.initials: M
authors.lastname: Lau
authors.affiliation: Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, MI, 48109-0632, United States. Electronic address: osawa@umich.edu.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Osawa
mesh_terms: D000818:Animals; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D006420:Hemeproteins; D011994:Recombinant Proteins; C496320:NOS1 protein, human; D052248:Nitric Oxide Synthase Type I
keywords: Bacterial expression system; Baculovirus expression system; Hemeprotein; Heterodimer; Homodimer; Nitric-oxide synthase; Site-directed mutagenesis