RESUMEN
The production of a homogeneous protein sample in sufficient quantities is an essential prerequisite not only for structural investigations but represents also a rate-limiting step for many functional studies. In the cell, a large fraction of eukaryotic proteins exists as large multicomponent assemblies with many subunits, which act in concert to catalyze specific activities. Many of these complexes cannot be obtained from endogenous source material, so recombinant expression and reconstitution are then required to overcome this bottleneck. This chapter describes current strategies and protocols for the efficient production of multiprotein complexes in large quantities and of high quality, using the baculovirus/insect cell expression system.
Asunto(s)
Baculoviridae/genética , Clonación Molecular/métodos , Insectos/virología , Complejos Multiproteicos/biosíntesis , Animales , Baculoviridae/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Insectos/citología , Insectos/genética , Complejos Multiproteicos/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.