Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae.

Abstract

Saccharomyces cerevisiae cells were transformed with plasmids containing hybrid genes in which the sequence encoding mature human epidermal growth factor was joined to sequences encoding the leader region (preprosegment) of the precursor of the yeast mating pheromone afactor. These cells accurately process the hybrid protein and efficiently secrete authentic biologically active human epidermal growth factor into the medium. Previous studies on the expression of B-lactamase-proinsulin gene fusions in Escherichia coli showed that the /3-lactamase signal sequence is cleaved and proinsulin is secreted into the periplasm (4). We reasoned, therefore, that the leader sequences of secreted yeast proteins may allow more efficient processing and secretion of heterologous proteins by yeast.Most studies on secretion of yeast proteins have used enzymes such as invertase and acid phosphatase (2, 5, 6), which are secreted into the periplasmic space or cell wall.We have used the peptide mating pheromone a-factor, which is efficiently secreted into the medium. a-Factor is a 13-residue peptide, secreted by cells of the a mating type, that acts on cells of the opposite a mating type to promote efficient conjugation between the two cell types leading to the formation of aa diploid cells (7). Studies on the sequence of the afactor structural gene (8) and on the synthesis and processing of the a-factor peptide (9-11) have shown that a-factor is synthesized as a precursor of 165 amino acids containing an 83-residue leader and four a-factor coding regions, each preceded by a short spacer peptide. The leader and spacer amino acids appear to contain the signals necessary for proteolytic processing and secretion. A recent study (12) shows that fusion of the leader region of the a-factor precursor to invertase, another secreted yeast protein, directs its export.To investigate whether the a-factor leader sequences are sufficient to allow efficient processing and secretion, we have constructed plasmids in which the genes coding for foreign secreted proteins have been fused to the yeast a-factor gene and expressed under the control of the a-factor promoter. Several different constructions were made in which the

Demonstration of Lysosomal Localization for the Mammalian Ependymin-related Protein Using Classical Approaches Combined with a Novel Density Shift Method

“…Both constructs contained a Myc epitope tag followed by a hexahistidine sequence at the C terminus (Myc-His) but differed in the presence or absence of a Glu-Ala-Glu-Ala sequence following an amino-terminal ␣ factor signal sequence. This Glu-Ala repeat may promote more efficient signal sequence cleavage under certain circumstances (29). After pilot experiments, we chose to use the construct lacking the Glu-Ala repeat for subsequent protein production, because expression levels from both constructs were similar, but N-terminal sequence analysis (data not shown) revealed that the Glu-Ala repeat was retained.…”

Immunodominant epitopes defined by a yeast-expressed library of random fragments of the rabies virus glycoprotein map outside major antigenic sites

“…The yeast expression system of a random peptide library (Brake et al, 1984) seemed particularly suitable for this study; in this method, peptides are secreted into the culture medium and are therefore directly accessible to the antibody without solubilization of the cells. Furthermore, Klepfer et al (1993) demonstrated that yeast was able to translate the rabies virus G gene as a 68 kDa protein immunoreactive not only with a polyclonal antiserum but also with three neutralizing MAbs that were known to react only with native protein, indicating that the yeast was probably able to efficiently process this protein.…”

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

“…6,1986 on May 10, 2018 by guest http://mcb.asm.org/ Downloaded from independent of the other side of the junction for this particular secretory system. Further experiments may elucidate these possibilities.…”

“…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.…”