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  • Title: New plasmid vectors for high level synthesis of eukaryotic fusion proteins in Escherichia coli.
    Author: Rimm DL, Pollard TD.
    Journal: Gene; 1989 Feb 20; 75(2):323-7. PubMed ID: 2653968.
    Abstract:
    Production of eukaryotic proteins in Escherichia coli has become rather simple since commercially available bacteriophage and plasmid vector systems allow investigators to select the optimal system for their particular problem. A common question is which system to use to produce the largest quantity of soluble recombinant protein with minimal, if any, bacterial protein fused to it. We have constructed a new set of plasmid vectors that produce large amounts of a fusion proteins that contain less than 25 amino acids of bacterial protein. We started with pATH-1, a plasmid expression vector comprised of the trpEp promoter and 37 kDa of the TrpE protein followed by a M13mp13 multiple cloning site for insertion of sequences to be expressed. We deleted the majority of the eukaryotic trpE sequence to produce a multiple frame, multiple enzyme cloning site, plasmid expression vector set called pRX. Transformation of E. coli CAG-456 (Baker et al., 1984) with this vector with an Acanthamoeba myosin tail sequence inserted in the correct frame yields a fusion protein that represents 45% of the total soluble protein. We have produced and purified 100 mg of this Acanthamoeba myosin-II fusion protein per liter of cell suspension.
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