New plasmid vectors for high level synthesis of eukaryotic fusion proteins in Escherichia coli.
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.
Rimm
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Department of Cell Biology and Anatomy, Johns Hopkins School of Medicine, Baltimore, MD 21205.
Pollard
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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
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D009218:Myosins