title: An efficient and generic strategy for producing soluble human proteins and domains in E. coli by screening construct libraries.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University, S-106 91 Stockholm, Sweden.
authors.forename: Tobias
authors.initials: T
authors.lastname: Cornvik
authors.forename: Sue-Li
authors.initials: SL
authors.lastname: Dahlroth
authors.forename: Audur
authors.initials: A
authors.lastname: Magnusdottir
authors.forename: Susanne
authors.initials: S
authors.lastname: Flodin
authors.forename: Benita
authors.initials: B
authors.lastname: Engvall
authors.forename: Victoria
authors.initials: V
authors.lastname: Lieu
authors.forename: Monica
authors.initials: M
authors.lastname: Ekberg
authors.forename: Pär
authors.initials: P
authors.lastname: Nordlund
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
chemical_list: D011994:Recombinant Proteins