Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells.
J Struct Biol
; 175(2): 198-208, 2011 Aug.
Article
en En
| MEDLINE
| ID: mdl-21419851
ABSTRACT
Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Proteínas Recombinantes
/
Clonación Molecular
/
Complejos Multiproteicos
/
Automatización de Laboratorios
Límite:
Animals
/
Humans
País/Región como asunto:
Europa
Idioma:
En
Revista:
J Struct Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2011
Tipo del documento:
Article
País de afiliación:
Francia