RESUMEN
We have designed and evaluated a novel strategy for screening large gene collections available as GATEWAY-adapted ORFeomes for soluble recombinant overexpression in Escherichia coli, called "Screening Colonies of ORFeome Pools" (SCOOP). From a large gene collection we could, without expensive multi-well based cloning and expression screening, determine which targets were suitable for large-scale expression and purification. Normalized bacterial overnight cultures of an ORF collection of entry clones derived from the Kaposi's sarcoma associated herpesvirus (KSHV) were pooled and used for the isolation of plasmid DNA. The resulting ORF library was subcloned into a prokaryotic expression vector in a single recombination reaction and was subsequently screened with the colony filtration (CoFi) blot for soluble recombinant overexpression in E. coli. ORFs determined to express soluble recombinant proteins were identified by sequencing and analysed by small-scale IMAC and SDS-PAGE. As a reference, we subcloned all ORFs individually using a traditional multi-well based procedure and screened them for soluble expression. Our results show that the two processes have a similar efficiency as 23 and 25 out of 74 assessable clones were identified as soluble expressers using SCOOP and the traditional multi-well procedure, respectively. Because SCOOP minimises costs for cloning and expression screening, it constitutes an interesting alternative for establishing expression of large gene collections. SCOOP also allows affordable screening in alternative vectors, expression strains and physical conditions, which is challenging in large-scale protein production programs. With this strategy in hand success rates for future proteome-wide protein production efforts can be significantly increased.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Genómica/métodos , Sistemas de Lectura Abierta , Biblioteca de Genes , Genómica/economía , Herpesvirus Humano 8/genética , Tamaño de la MuestraRESUMEN
The implementation of generic and efficient technologies for the production of recombinant eukaryotic proteins remains an outstanding challenge in structural genomics programs. We have recently developed a new method for rapid identification of soluble protein expression in E. coli, the colony filtration blot (CoFi blot). In this study, the CoFi blot was used to screen libraries where the N-terminal translation start point was randomized. To investigate the efficiency of this strategy, we have attributed a large number of proteins to this process. In a set of 32 mammalian proteins, we were able to double the success rate (from 34 to 68%) of producing soluble and readily purifiable proteins in E. coli. Most of the selected constructs had their N-termini close to predicted domain borders and the method therefore provides a mean for experimental "domain foot printing." Surprisingly, for most of the targets, we also observed expressing constructs that were close to full-length. In summary this strategy constitutes a generic and efficient method for producing mammalian proteins for structural and functional studies.