RESUMEN
Lynx1 expresses in the central nervous system and plays important role in a regulation of nicotinic acetylcholine receptors. Successful milligram-quantitive expression of ws-Lynx1 was achieved only in the case of its production in the form of cytoplasm inclusion bodies. Different conditions of ws-Lynx1 refolding for yield optimization were performed. The obtained recombinant protein was characterized by means of mass spectrometry and CD spectroscopy. The binding experiments on the nAChRs from Torpedo californica membranes revealed that ws-Lynxl is biologically active and blocks muscle nAChR with IC50-20-30 microM.
Asunto(s)
Proteínas Ligadas a GPI/biosíntesis , Neurotransmisores/biosíntesis , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bungarotoxinas/antagonistas & inhibidores , Órgano Eléctrico/química , Órgano Eléctrico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/farmacología , Expresión Génica , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Neurotransmisores/química , Neurotransmisores/farmacología , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Solubilidad , TorpedoRESUMEN
The cell-free expression system based on bacterial extract S30 from E. coli for production of the transmembrane domain of human receptor tyrosine kinase ErbB3 (residues 632-675) was developed. The synthesis of the domain in the soluble form in the presence of detergents and in the form of the translation mixture precipitate was studied. The protocols of purification of the recombinant domain obtained by both methods were developed. The final yield of target protein in optimal conditions was 1.8-2.0 mg per 1 ml of translation mixture.
Asunto(s)
Ingeniería de Proteínas/métodos , Receptor ErbB-3/química , Proteínas Recombinantes/química , Sistema Libre de Células , Cromatografía de Afinidad , Detergentes/química , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Membranas Artificiales , Plásmidos , Receptor ErbB-3/genética , Proteínas Recombinantes/genética , Solubilidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
alpha-Neurotoxins from snake venom are highly efficient inhibitors of nicotinic acetylcholine receptors (nAChR). These small proteins that have a beta-structural organization attract much interest as a tool for studies of nACh R and as prototypes for the development of new Pharmaceuticals for the treatment of diseases of the nervous system. However, the in vitro production of "three-finger" neurotoxins is complicated by the presence of four or five disulfide bonds that are closely located in their molecules. The present review contains a description of the most frequently used modern approaches for the E. coli expression of recombinant proteins (direct expression, expression as fusions, and secretion) with an emphasis placed on the recombinant production of snake alpha-neurotoxins. The methods of E. coli expression of isotopically labeled neurotoxins are described. The proposed solutions will be of broad interest for the bacterial production of other disulfide-abundant proteins.
Asunto(s)
Disulfuros/química , Neurotoxinas/biosíntesis , Venenos de Serpiente/química , Secuencia de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Marcaje Isotópico , Datos de Secuencia Molecular , Neurotoxinas/química , Neurotoxinas/genética , Biosíntesis de Péptidos , Péptidos/síntesis química , Conformación Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Neurotoxin II from the venom of cobra Naja oxiana is a short type alpha-neurotoxin, which competitively inhibits nicotinic acetylcholine receptor. The toxin gene was expressed as a construct fused with the thioredoxin gene and the linker encoding the enteropeptidase recognition site and a Met residue between the genes. The fusion protein was mainly cleaved by cyanogen bromide, since enteropeptidase was less effective. The yield of neurotoxin II was 6 mg/l of the bacterial culture. The resulting recombinant protein was identified with native neurotoxin II by its N-terminal analysis, mass spectrometry, and NMR spectroscopy. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.