title: [Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family members].
abstract: A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.
authors.forename: M V
authors.initials: MV
authors.lastname: Goncharuk
authors.forename: A A
authors.initials: AA
authors.lastname: Shul'ga
authors.forename: Ia S
authors.initials: IaS
authors.lastname: Ermoliuk
authors.forename: E N
authors.initials: EN
authors.lastname: Tkach
authors.forename: S A
authors.initials: SA
authors.lastname: Goncharuk
authors.forename: Iu E
authors.initials: IuE
authors.lastname: Pustovalova
authors.forename: K S
authors.initials: KS
authors.lastname: Mineev
authors.forename: É V
authors.initials: ÉV
authors.lastname: Bocharov
authors.forename: I V
authors.initials: IV
authors.lastname: Maslennikov
authors.forename: A S
authors.initials: AS
authors.lastname: Arsen'ev
authors.forename: M P
authors.initials: MP
authors.lastname: Kirpichnikov
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020893:Receptor, ErbB-3 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D012995:Solubility; D014169:Transformation, Bacterial
chemical_list: D008823:Micelles; D010455:Peptides; D020033:Protein Isoforms; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; D011993:Recombinant Fusion Proteins