RESUMO
The potent activator and chemoattractant for human neutrophils, neutrophil-activating peptide 2 (NAP-2), has been cloned and expressed in Escherichia coli. The protein has been purified to homogeneity (> 98%) by a series of chromatographic techniques, including reversed phase HPLC. The biological activity of recombinant human NAP-2 (rhNAP-2), characterized by the induction of elastase release from human neutrophils, was found to be comparable to natural NAP-2. rhNAP-2 has been crystallized by the hanging drop vapor diffusion method. The crystals belong to space group P222 with unit cell dimensions of a = 30.8 A, b = 39.5 A and c = 95.3 A. A packing density of 3.8 A3/Da with a solvent content of approximately 68% is obtained when one molecule per asymmetric unit is assumed. The crystals were shown to diffract to beyond 2.0 A on a conventional X-ray source. They are stable to X-rays for several days and are thus suitable for high resolution structure determination.
Assuntos
Cristalografia por Raios X , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Cristalização , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Expressão Gênica , Humanos , Elastase de Leucócito , Dados de Sequência Molecular , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Elastase Pancreática/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , beta-TromboglobulinaRESUMO
Phosphonate analogue 5 of the lipid A precursor 4 has been prepared from phosphonate 2 and nucleotide 3 with the help of lipid A synthase, isolated from the overproducing Escherichia coli mutant MC 1061 (delta 2512) or JB1104 (delta 2514). The biological properties of phosphonate 5 and phosphate 4 are quite similar to each other as compared in the limulus amoebocyte lysate assay, by the activation of the RAW264 murine macrophagelike cell line (determined by stimulation of ornithine decarboxylase), and by the pyrogenicity in rabbits. Hydrolytic removal of the 1-phosphate group of 4 is thus not a prerequisite for its biological activity.
Assuntos
Hexosiltransferases/metabolismo , Lipídeo A/análogos & derivados , N-Acetilglucosaminiltransferases , Compostos Organofosforados/síntese química , Precursores de Proteínas , Animais , Temperatura Corporal/efeitos dos fármacos , Escherichia coli/enzimologia , Teste do Limulus , Lipídeo A/síntese química , Lipídeo A/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Estrutura Molecular , Compostos Organofosforados/farmacologia , Ornitina Descarboxilase/metabolismo , Precursores de Proteínas/química , Coelhos , Células Tumorais CultivadasAssuntos
Escherichia coli/genética , Interleucina-3/biossíntese , Sequência de Aminoácidos , Clonagem Molecular , DNA Bacteriano/genética , Eletroforese em Gel de Poliacrilamida , Enteropeptidase/metabolismo , Humanos , Interleucina-3/genética , Interleucina-3/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
Interleukin-8 (neutrophil-activating factor; NAP-1) has been crystallized by the vapour diffusion technique to give single crystals suitable for three-dimensional structural study at a resolution higher than 2.4 A. The crystals belong to the space group P3(1)21 or P3(2)21 and have unit cell dimensions a = b = 40.9 A, c = 90.3 A.