RESUMEN
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.
Asunto(s)
Membrana Celular/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Péptidos/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Membrana Celular/genética , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Clonación Molecular , Dimerización , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Marcaje Isotópico , Micelas , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Péptidos/aislamiento & purificación , Plásmidos , Isoformas de Proteínas , Estructura Terciaria de Proteína , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-2/aislamiento & purificación , Receptor ErbB-3/química , Receptor ErbB-3/genética , Receptor ErbB-3/aislamiento & purificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Solubilidad , Transformación BacterianaRESUMEN
The C40,82A;I87E mutant of barstar, an intracellular inhibitor of the ribonuclease barnase from Bacillus amyloliquefaciens, was obtained, and its physicochemical properties were studied. It was produced as a fusion protein with thioredoxin and then cleaved from this by EKmax enterokinase. The mutant was shown by NMR to retain the spatial structure of the wild-type protein but, in contrast to barstar, does not form the homodimers characteristic of barstar in aqueous solution. The mutant protein binds barnase with the dissociation constant (6.6 +/- 1.1) x 10(-11) M and exhibits other physicochemical properties similar to those of the wild-type barstar. This allows the use of C40,82A;I87E mutant instead of wild-type barstar in investigations where the protein dimerization is undesirable. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.
