Screening colonies of pooled ORFeomes (SCOOP): a rapid and efficient strategy for expression screening ORFeomes in Escherichia coli.
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.
Dahlroth
Sue-Li
SL
Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden.
Lieu
Victoria
V
Haas
Jürgen
J
Nordlund
Pär
P
D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D023281:Genomics / Q000191:economics / Q000379:methods*; D019288:Herpesvirus 8, Human / Q000235:genetics; D016366:Open Reading Frames; D018401:Sample Size