Synthesis of a human insulin gene. VI. Expression of the synthetic proinsulin gene in yeast.
The construction of plasmid vectors for the controlled expression of a synthetic human proinsulin gene in the yeast Saccharomyces cerevisiae is described. Attempts to express the proinsulin gene using the yeast ADH1 promoter alone did not yield detectable levels of proinsulin. Successful expression was achieved when the proinsulin gene was fused with the promoter and protein leader sequence of the GAL1 gene (coding for yeast galactokinase) in the yeast-Escherichia coli plasmid vector pYT7810. Two different-length leader sequences were employed; the longer leader (about 280 amino acids, fusion plasmid pPS13) gave about five times greater expression than the shorter leader fusion (30 amino acids, plasmid pPS5). Both fusions gave soluble protein products, and the proinsulin could be cleaved by cyanogen bromide treatment from the leader polypeptide. Proinsulin was detected by radioimmunoassay for human C-peptide only in cells induced with galactose, and was not detected in the gene fusions that were out of phase with the GAL1 leader sequence. Methods of improving the level of expression of the proinsulin gene in yeast using this system are discussed.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D005686:Galactokinase / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D007328:Insulin / Q000235:genetics*; D011384:Proinsulin / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004274:DNA, Recombinant; D007328:Insulin; D011384:Proinsulin; D005686:Galactokinase