Optimization of recombinant protein expression level in Escherichia coli by flow cytometry and cell sorting.
Overexpression of recombinant proteins in Escherichia coli often leads to a severe growth retardation of the host cells. The phage T7 promoter phi10 in a pET vector was utilized to express human superoxide dismutase. Induction with IPTG lead to an increase in protein content and cell size and a termination of cell division, due to the deviation of the general metabolic fluxes from all cellular processes to plasmid maintenance and foreign protein synthesis. To generate promoter mutants which are better tolerated by the host cells we constructed a random mutation library by PCR with degenerated primers in a part of the promoter involved in the binding to the RNA polymerase and the initiation of transcription. This library was sorted by flow cytometry for cells with a lower total protein content as an indicator for continued cell replication and hence a less severe stress situation. The clones obtained had a similar SOD production compared to the original strain, but were able to reach higher densities in a batch culture, which resulted in a higher total yield.
Soriano
Elena
E
Institute of Applied Microbiology, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Borth
Nicole
N
Katinger
Hermann
H
Mattanovich
Diethard
D
D001426:Bacterial Proteins / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000145:classification / Q000235:genetics*; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005786:Gene Expression Regulation; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D015202:Protein Engineering / Q000295:instrumentation / Q000379:methods*; D011786:Quality Control; D011994:Recombinant Proteins / Q000235:genetics*; D011995:Recombination, Genetic; D013045:Species Specificity; D013482:Superoxide Dismutase / Q000235:genetics
D001426:Bacterial Proteins; D019151:Peptide Library; D011994:Recombinant Proteins; D013482:Superoxide Dismutase