Biochemistry a - Factor - directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae ( epidermal growth factor / protein fusion / peptide processing / yeast mating type / in vitro mutagenesis )

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. The secretion of proteins by the yeast Saccharomyces cerevisiae occurs by a mechanism very similar to that observed in mammalian cells. In studies using temperature-sensitive protein secretion (sec) mutants, Schekman and collaborators (1, 2) have shown that the yeast secretory pathway involves a series of membrane-bound structures that mediate the transfer of exported proteins from their site of synthesis at the endoplasmic reticulum to their site of release at the plasma membrane. As in other organisms, these secreted proteins are synthesized as larger precursors including "signal" sequences, which are cleaved by a membrane-bound protease to yield the mature gene products. Previously published studies on the secretion of heterologous proteins from yeast have used the signal sequences of the mammalian proteins being secreted (3). When a structural gene of a-interferon was expressed in yeast to produce a peptide containing the signal sequence, only 109-20% of the interferon synthesized was secreted into the medium. In addition, proteolytic processing seemed to be imprecise because a heterogeneous mixture of peptides was produced, apparently resulting from cleavages at several different sites within the interferon precursor (3). 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 DNA sequences of the spacer region, which may encode some of the processing recognition sites, were altered to study their role in the maturation process. Yeast cells transformed with some of these plasmids efficiently synthesize, process, and secrete into the medium the mature biologically active foreign proteins. We describe below the expression, processing, and secretion of human epidermal growth factor (hEGF) from a chemically synthesized gene using the yeast a-factor expression system. MATERIALS AND METHODS Strains. Plasmids were propagated in E. coli strain HB101 grown in LB medium (13). S. cerevisiae strain AB103 (Mata leu2-3,112 ura 3-52 his4-580 pep 4-3) was constructed in this laboratory. JRY188 (Mata sir3-8 leu2-3,112 ura 3-52 his4 rme) was obtained from Jasper Rine. Yeast transformation was carried out as described (14). Plasmids. Plasmid DNA was isolated by alkaline NaDodSO4 lysis (15). Plasmid pAB11 was derived from pBR322 (16) as described (10). pC1/1 was derived from pJDB219 (17) by replacement of the pMB9 region by pBR322 and contains the ampicillinand tetracycline-resistance genes and replication origin of pBR322 for selection and propagation of E. coli and the yeast LEU2 gene and 2-gm plasmid sequences for selection and autonomous replication in yeast leu2 mutants. An oligonucleotide (5'-T-T-A-G-T-A-C-A-T-T-G-G-T-T-GG-C-C-G-G-3') homologous to the end of the yeast a-factor structural gene (8) was used to probe a library (6) of yeast genomic DNA cloned in YEP24 (18). A plasmid (pAB101; see ref. 10) with a 7.5-kilobase-pair (kbp) insert was isolated from which was obtained a 1.76-kbp EcoRI fragment containing the complete yeast a-factor gene. This fragment was cloned into pABil to produce pAB112. In Vitro Mutagenesis. Site-directed in vitro mutagenesis (19) was performed using single-stranded DNA from phage M13 into which had been cloned a 421-base-pair (bp) Pst I/Sal I fragment containing the a-factor leader-hEGF gene fusion. A synthetic oligonucleotide (70 pmol) was used as a primer for the synthesis of the second strand from 1.2 pmol of the phage template using the Klenow fragment of DNA polymerase I (Bethesda Research Laboratories; 5 units) and Abbreviations: hEGF, human epidermal growth factor; kbp, kilobase pair(s); bp, base pair(s). 4642 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. NatL Acadl Sci USA 81 (1984) 4643 T4 DNA ligase (New England Biolabs; 40 units). After 18 hr at 14'C, the mixture was treated with S1 nuclease (Miles; 5 units) for 15 min and was used' to transfect E. coli JM101 cells. Bacteriophage containing the desired' mutation were located by plaque-filter hybridization using the 32P-labeled primer as a probe. EGF Assays. EGF was assayed by a competitive receptor binding assay using I251-labeled mouse EGF (Amersham) and human foreskin fibroblasts (20). Standard curves were obtained by measuring the effects of increasing quantities of mouse EGF on the binding of a constant amount of I251-labeled mouse EGF.