Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 3 de 3
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Struct Biol ; 177(2): 329-34, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22245778

RESUMO

Structural biology studies typically require large quantities of pure, soluble protein. Currently the most widely-used method for obtaining such protein involves the use of bioinformatics and experimental methods to design constructs of the target, which are cloned and expressed. Recently an alternative approach has emerged, which involves random fragmentation of the gene of interest and screening for well-expressing fragments. Here we describe the application of one such fragmentation method, combinatorial domain hunting (CDH), to a target which historically was difficult to express, human MEK-1. We show how CDH was used to identify a fragment which covers the kinase domain of MEK-1 and which expresses and crystallizes significantly better than designed expression constructs, and we report the crystal structure of this fragment which explains some of its superior properties. Gene fragmentation methods, such as CDH, thus hold great promise for tackling difficult-to-express target proteins.


Assuntos
MAP Quinase Quinase 1/química , MAP Quinase Quinase 1/genética , Engenharia de Proteínas , Clonagem Molecular , Cristalização , Cristalografia , Escherichia coli , Humanos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
2.
J Gen Virol ; 85(Pt 4): 821-831, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15039525

RESUMO

Infection of insect larvae with Autographa californica nucleopolyhedrovirus (AcMNPV) results in the liquefaction of the host, a process involving the action of virus-encoded chitinase and cathepsin gene products. Chitinase is localized in the endoplasmic reticulum (ER) during infection because of the presence of a C-terminal ER retrieval motif (KDEL). In this study, the KDEL coding region was removed from the chitinase gene so that expression of the modified chitinase remained under the control of its own gene promoter, at its native locus. The deletion of KDEL resulted in the redistribution of chitinase within the cell during virus infection. Chitinase lacking the KDEL motif was detectable at the plasma membrane and was also evident in the culture medium of virus-infected cells from as early as 12 h post-infection (p.i.). Secretion of chitinase from the cell continued up to 72 h p.i., until cytolysis. The biological activity of the recombinant virus in Trichoplusia ni larvae was enhanced, with a significant reduction in the lethal dose and lethal time associated with infection. Furthermore, a reduction in feeding damage caused by infected larvae was observed compared to AcMNPV-infected individuals.


Assuntos
Quitinases/genética , Nucleopoliedrovírus/enzimologia , Nucleopoliedrovírus/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Quitinases/química , Quitinases/metabolismo , DNA Viral/genética , Genoma Viral , Dados de Sequência Molecular , Mariposas , Nucleopoliedrovírus/patogenicidade , Oligopeptídeos , Sinais Direcionadores de Proteínas , Deleção de Sequência , Spodoptera , Virulência
3.
J Gen Virol ; 83(Pt 3): 685-694, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11842263

RESUMO

During virus infection of insect cells, the Autographa californica nucleopolyhedrovirus chitinase is localized primarily within the endoplasmic reticulum (ER), which is consistent with the presence of a carboxy-terminal ER retention motif (KDEL). Release of chitinase into the extracellular medium appears to be concomitant with terminal cell lysis, rather than by active secretion. In this study, we have shown that mutation of the KDEL motif induces a partial redistribution of the chitinase at both early and late times post-infection. Deletion of the KDEL motif or substitution with glycine residues allowed chitinase to move through the secretory pathway, accumulating to detectable levels in the extracellular medium by 24 h post-infection; more than 48 h prior to cell lysis. Deletion of the KDEL motif did not compromise enzyme activity, with the modified enzyme exhibiting characteristic endo- and exo-chitinolytic activity. Trichoplusia ni larvae infected with the modified virus were found to liquefy approximately 24 h earlier than larvae infected with a control virus in which the chitinase KDEL motif had not been deleted.


Assuntos
Quitinases/química , Quitinases/metabolismo , Retículo Endoplasmático/metabolismo , Nucleopoliedrovírus/enzimologia , Nucleopoliedrovírus/genética , Oligopeptídeos/genética , Sinais Direcionadores de Proteínas/genética , Deleção de Sequência/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Baculoviridae/genética , Sequência de Bases , Linhagem Celular , Quitinases/biossíntese , Quitinases/genética , Cisteína Endopeptidases/metabolismo , Retículo Endoplasmático/virologia , Larva/crescimento & desenvolvimento , Larva/virologia , Lepidópteros/crescimento & desenvolvimento , Lepidópteros/virologia , Dados de Sequência Molecular , Nucleopoliedrovírus/patogenicidade , Nucleopoliedrovírus/fisiologia , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Taxa de Sobrevida , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...