Construction of a high-copy "ATG vector" for expression in Escherichia coli.
We report the construction of an inducible, high-copy plasmid for the expression of foreign proteins in Escherichia coli. This plasmid, pPB1, combines the trc promoter, beta-galactosidase translation start site, and polylinker of pKK233-2 with the origin of replication region of pUC19. Replacement of the origin of replication of pKK233-2 results in a threefold increase in plasmid copy number of pPB1 compared with pKK233-2. Subclones of the cDNA for rabbit muscle fructose-1,6-bisphosphate aldolase (E.C. 4.1.2.13) in the two expression plasmids exhibit a comparable difference in copy number. An increase in protein expression measured by SDS-PAGE and aldolase specific activities reflects the increased copy number. Specific activities of aldolases in bacterial extracts differ approximately sixfold between the two expression plasmids in E. coli JM83. Aldolase A can compose up to 40% of the total protein in E. coli JM83 when expressed in pPB1, from which more than 100 mg of purified enzyme can be obtained per liter culture.
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Biology Department, Boston University, Massachusetts 02215.
Tolan
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D001483:Base Sequence; D004261:DNA Replication / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005634:Fructose-Bisphosphate Aldolase / Q000096:biosynthesis / Q000302:isolation & purification; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D011994:Recombinant Proteins; D005634:Fructose-Bisphosphate Aldolase