Mogens Trier Hansen

Secretion of human insulin by a transformed yeast cell

A yeast expression plasmid encoding a mini‐proinsulin molecule was constructed and transformed into Saccharomyces cerevisiae. The plasmid encoded the sequence: B‐Arg‐Arg‐Leu‐Gln‐Lys‐Arg‐A in which B represents the B‐chain (30 amino acid residues) and A represents the A‐chain (21 amino acid residues) of human insulin. The secreted peptides were shown to be a mixture of human insulin and des(B‐30)human insulin. Thus, correct disulphide bridges can be established in proinsulin‐like molecules devoid of a normal C‐peptide region. Furthermore, the specificity of the yeast processing enzymes is so similar to the proinsulin converting enzymes in the human pancreatic β‐cell that it allows the processing of the mini‐proinsulin to insulin.

The secretion of glucagon by transformed yeast strains

Saccharomyces cerevisiae strains were transformed with plasmids coding for modified mating factor α1 leader sequences followed by glucagon. Glucagon‐containing peptides which were secreted into the fermentation broth were isolated and their amino acid sequences determined. The yeast strain transformed with the sequence coding for the complete mating factor α1 leader sequence preceding the glucagon gene (MT556) secreted glucagon plus glucagon extended at its N‐terminal by parts of the leader sequence. The yeast strain transformed with the sequence coding for a truncated mating factor α1 leader sequence before the glucagon gene (MT615) secreted glucagon. These observations suggest that S. cerevisiae is a suitable vehicle for the efficient expression of plasmids coding for polypeptides similar to glucagon (e.g. VIP, secretin, GIP).

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.