Secretion of foreign proteins from Saccharomyces cerevisiae directed by alpha-factor gene fusions.

Abstract

Fusions between the cloned yeast a-factor structural gene and chemically synthesized DNA segments encoding human protein analogs have been constructed. The gene fusions encode hybrid proteins that include the first 89 amino acids of the native a-factor precursor fused to either a small (j8-endorphin, 31 amino acids) or large (a-interferon, 166 amino acids) foreign protein. Proteolytic cleavage sites involved in a-factor maturation from the native precursor immediately precede the foreign peptide in the hybrid protein.The a-factor promoter was utilized to express the gene fusions in Saccharomyces cerevisiae and resulted in the efficient secretion of the foreign proteins into the culture medium. The processing of the hybrid proteins has been characterized by amino acid sequence analysis of the secreted proteins. The proteolytic cleavages involved in the maturation of a-factor peptides from the native precursor also occur accurately in the hybrid protein. In addition, cleavages occurred on the carboxyl side of two lysines within the .6-endorphin peptide. Internal cleavages in the interferon protein were also detected. However, in this case, the cleavages occurred at a very low frequency such that >95% of the secreted interferon remained intact.

Expression, assembly and secretion of a fully active plant ferredoxin‐NADP+ reductase by Saccharomyces cerevisiae

“…Since we have examined polypeptides secreted through the cell wall to the culture medium we cannot eliminate the possibility that processing has occurred inside the ER or after the protein has reached the outside. The products of genes KEX2 [31] and STE13 [21] have been identified as responsible for a-pheromone maturation (Fig. 2).…”

“…However, secretion in minimal medium was undetectable throughout the range of temperatures tested (data not shown). Solid arrows show the processing site recognized by the KEX2 gene product [31] and open arrows those for the STE13 product [21]. The natural ferredoxin-NADP' reductase transit peptide: may favor secretion by precluding enzyme folding or by facilitating unfolding after translation.…”

Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides.

“…Recently there have been several reports of secretion of heterologous protein products by using the a-factor gene of yeast cells (4,6,26). These systems result in proper processing of heterologous proteins during the secretion process.…”

Construction of a yeast vector directing the synthesis and release of barley (1→3, 1→4)-β-glucanase

“…Attempts to obtain secretion of other heterologous proteins have mainly focussed on fusion of the gene with a signal peptide coding region from yeast, e.g. the yeast ct-factor gene (6,8,34) and the gene fragment encoding the signal peptide code of the secreted form of yeast invertase (11). JIGAM1 et al (21) synthesized a DNA fragment encoding the signal peptide of chicken lysozyme and fused that to a synthetic human lysozyme gene.…”