Competitive expression of two heterologous genes inserted into one plasmid in Saccharomyces cerevisiae.
Plasmids were constructed which contained two expression units encoding single-chain insulin precursors. Surprisingly, the total amount of insulin precursor produced was similar to that produced from plasmids containing a single expression unit. In this system, therefore, two expression cassettes can be brought to compete for the limited ability of the yeast cell for synthesis and secretion. Using genes encoding B(1-29)-A(1-21) and B(1-29)-Ala-Ala-Lys-A-(1-21), the slightly different precursors could be quantified individually after separation by high-performance liquid chromatography from the culture supernatant. The two-cassette system allowed a sensitive and well controlled comparison of parameters important for optimal expression of a heterologous gene in Saccharomyces cerevisiae. The system was used to compare two promoter constructions and also to evaluate the position of expression cassettes in the plasmid. Finally the codon usage in the gene to be expressed was found to influence its ability to compete for expression.
Egel-Mitani
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M
Novo Research Institute, Bagsyaerd, Denmark.
Hansen
M T
MT
Norris
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K
Snel
L
L
Fiil
N P
NP
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002851:Chromatography, High Pressure Liquid; D003062:Codon; D005796:Genes; D006801:Humans; D007328:Insulin / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013728:Terminator Regions, Genetic
D003062:Codon; D007328:Insulin; D011994:Recombinant Proteins