title: Using an Escherichia coli cell-free extract to screen for soluble expression of recombinant proteins.
abstract: For structural and functional genomics programs, new high-throughput methods to characterize well-expressing and highly soluble proteins are essential. A faster and more convenient approach to screen expression conditions of recombinant proteins compared to classical in vivo systems is the Escherichia coli cell-free expression system. Here, we describe a rapid procedure to screen for expression and solubility of recombinant proteins using an E. coli cell-free extract. The results presented cover 24 open reading frames of unknown function from different micro-organisms. In order to screen different variables that may interfere with solubility, we expressed the recombinant proteins with a histidine6 tag, either N-terminal or C-terminal at two temperatures (25 degrees C and 30 degrees C). The identification of recombinant proteins is performed by the dot blot procedure using an anti-histidine tag antibody. We designed a rapid method that allows the characterization of soluble candidates from a large number of genes or from a large number of variants that is highly compatible with structural genomics expectations.
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720-5230, USA.
authors.forename: Didier
authors.initials: D
authors.lastname: Busso
authors.forename: Rosalind
authors.initials: R
authors.lastname: Kim
authors.forename: Sung-Hou
authors.initials: SH
authors.lastname: Kim
mesh_terms: D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D023281:Genomics; D016366:Open Reading Frames; D040901:Proteomics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D012995:Solubility; D014170:Transformation, Genetic
chemical_list: D004274:DNA, Recombinant; D011994:Recombinant Proteins