Balancing the production of two recombinant proteins in Escherichia coli by manipulating plasmid copy number: high-level expression of heterodimeric Ras farnesyltransferase.
The native Ras farnesyltransferase heterodimer (alpha beta) and a heterodimer with a truncated alpha subunit (alpha' beta) were overproduced at a high level and in a soluble form in Escherichia coli. The alpha, alpha', and beta subunits were synthesized from individual plasmid vectors under the control of bacteriophage T7 promoters. Although each subunit could be expressed at a high level by itself, when either the alpha or alpha' and the beta plasmid were present in cells at the same time, the alpha and alpha' subunits were preferentially expressed to such a degree that little or none of the beta subunit accumulated. A satisfactory balance between both combinations of subunits (alpha beta and alpha' beta) was achieved by making incremental adjustments in the copy number of the beta-encoding plasmid. As the copy number of the beta plasmid increased, so did the ratio of beta:alpha or beta:alpha', but there was little difference in the total amount of recombinant protein (alpha + beta or alpha' + beta) that was produced. This may be a generally useful method for balancing the production of two recombinant polypeptides in E. coli. A noteworthy advantage of this approach is that it can be undertaken without first determining the cause of the imbalance.
Tsao
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KL
Department of Physical Chemistry, Roche Research Center, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110, USA.
Waugh
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DS
D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D017123:Bacteriophage T7; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D017931:DNA Primers; D046911:Macromolecular Substances; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; C085706:FLAG peptide; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase