title: Use of GFP fusions for the isolation of Escherichia coli strains for improved production of different target recombinant proteins.
abstract: High level expression of a recombinant gene results in growth arrest, followed by overgrowth by non-productive derivatives. Two methods are described for the isolation of E. coli BL21* strains that are improved hosts for recombinant protein production. Both are based upon the observations (i) that fluorescence of a C-terminal GFP tag is a reliable reporter of the production and correct folding of the N-terminal target domain; and (ii) rare mutants arise spontaneously that remain productive during long periods of high level recombinant protein production. The first method relies upon identifying these mutants amongst colonies on agar plates; the other exploits fluorescence activated cell sorting. Although identical mutations in the regulatory region of the T7 polymerase gene were found in all of the improved host strains isolated, they differed in their ability to accumulate the outer membrane protein, Ccp, or a cytoplasmic protein, CheY-GFP. Cytochrome c peroxidase activity of recombinant Ccp from one of these strains was demonstrated. Changes in levels of T7 polymerase expression are therefore insufficient to ensure increased accumulation of all recombinant proteins. We demonstrate that the methods described allow strains to be isolated that carry other, currently uncharacterised mutations that are required depending on the target protein.
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
authors.forename: Sara
authors.initials: S
authors.lastname: Alfasi
authors.forename: Yanina
authors.initials: Y
authors.lastname: Sevastsyanovich
authors.forename: Lenny
authors.initials: L
authors.lastname: Zaffaroni
authors.forename: Lesley
authors.initials: L
authors.lastname: Griffiths
authors.forename: Richard
authors.initials: R
authors.lastname: Hall
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Cole
mesh_terms: D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D015169:Colony Count, Microbial; D003578:Cytochrome-c Peroxidase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D013312:Stress, Physiological / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D011993:Recombinant Fusion Proteins; C000607606:cheY protein, E coli; D049452:Green Fluorescent Proteins; D003578:Cytochrome-c Peroxidase