title: New plasmid vectors for high level synthesis of eukaryotic fusion proteins in Escherichia coli.
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.
authors.affiliation: Department of Cell Biology and Anatomy, Johns Hopkins School of Medicine, Baltimore, MD 21205.
authors.forename: D L
authors.initials: DL
authors.lastname: Rimm
authors.forename: T D
authors.initials: TD
authors.lastname: Pollard
mesh_terms: D000048:Acanthamoeba / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D005057:Eukaryotic Cells / Q000648:ultrastructure; D005784:Gene Amplification; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009218:Myosins / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping
chemical_list: D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D009218:Myosins