RESUMO
The four core histone proteins, H2A, H2B, H3, and H4 of Xenopus laevis have been individually expressed in milligram quantities in Escherichia coli. The full-length proteins and the "trypsin-resistant" globular domains were purified under denaturing conditions and folded into histone octamers. Both intact and truncated recombinant octamers, as well as chicken erythrocyte octamer, were assembled into nucleosome core particles using a 146 bp defined-sequence DNA fragment from a 5 S RNA gene. The three types of core particles were characterized and compared by gel electrophoresis, DNase I cleavage, and tyrosine fluorescence emission during stepwise dissociation with increasing ionic strength. Nucleosome core particles containing native and mutant histones made in bacteria have facilitated its X-ray structure determination at 2.8 A resolution.
Assuntos
Histonas/biossíntese , Nucleossomos/química , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Animais , Galinhas , Clonagem Molecular , Eritrócitos/química , Escherichia coli/genética , Genes Sintéticos , Histonas/genética , Histonas/isolamento & purificação , Dados de Sequência Molecular , Plasmídeos/genética , Ligação Proteica , Dobramento de Proteína , RNA Ribossômico 5S , Proteínas Recombinantes/isolamento & purificação , Xenopus laevis/genéticaRESUMO
The endoglycosidase peptide: N-glycosidase, secreted by the Gram-negative bacterium Flavobacterium meningosepticum (PNGase F), has been isolated, purified to homogeneity and crystallized from polyethylene glycol solutions using vapour diffusion and seeding techniques. The crystals are orthorhombic, space group P2(1)2(1)2, with unit cell dimensions a = 85.07 A, b = 85.14 A, c = 48.50 A, and are suitable for high resolution X-ray structure analysis.