title: Effective isotope labeling of proteins in a mammalian expression system.
abstract: Isotope labeling of biologically interesting proteins is a prerequisite for structural and dynamics studies by NMR spectroscopy. Many of these proteins require mammalian cofactors, chaperons, or posttranslational modifications such as myristoylation, glypiation, disulfide bond formation, or N- or O-linked glycosylation; and mammalian cells have the necessary machinery to produce them in their functional forms. Here, we describe recent advances in mammalian expression, including an efficient adenoviral vector-based system, for the production of isotopically labeled proteins. This system enables expression of mammalian proteins and their complexes, including proteins that require posttranslational modifications. We describe a roadmap to produce isotopically labeled (15)N and (13)C posttranslationally modified proteins, such as the outer domain of HIV-1 gp120, which has four disulfide bonds and 15 potential sites of N-linked glycosylation. These methods should allow NMR spectroscopic analysis of the structure and function of posttranslationally modified and secreted, cytoplasmic, or membrane-bound proteins.
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. Electronic address: sastrym@niaid.nih.gov.
authors.forename: Mallika
authors.initials: M
authors.lastname: Sastry
authors.affiliation: Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA. Electronic address: CaroleB@mail.nih.gov.
authors.forename: Carole A
authors.initials: CA
authors.lastname: Bewley
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. Electronic address: pdkwong@nih.gov.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Kwong
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D008322:Mammals / Q000378:metabolism*; D011506:Proteins / Q000235:genetics*
chemical_list: D011506:Proteins
keywords: Adenovirus vector; Glycoproteins; Isotope labeling; Mammalian expression