Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reissue Patent
1998-08-27
2001-08-28
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069700, C435S069800, C435S254200, C435S254110, C435S320100, C435S471000, C435S483000, C536S024100, C536S024200, C536S023400
Reissue Patent
active
RE037343
ABSTRACT:
Related U.S. Application Data
This is a reissue of U.S. Pat. No. 5,602,034, Issued, Feb. 11, 1997, incorporated herein by reference in its entirety.
TECHNICAL FIELD
The present invention relates to the production of recombinant proteins in yeast. More particularly, the present invention is directed to an improved &agr;-factor expression system which provides for the secretion of heterologous proteins from yeast host cells.
BACKGROUND
Kurjan et al. (1982) Cell 30:933-943 discloses the first cloning and sequencing of a gene encoding a yeast &agr;-factor precursor gene. Kurjan et al., U.S. Pat. No. 4,546,082, also reports the cloning of this gene, and suggests that the &agr;-factor leader sequence can be employed to direct the secretion of heterologous proteins in yeast. The patent, however, does not contain data which would indicate that the patentees ever successfully employed the &agr;-factor leader to express and secrete a heterologous protein in yeast.
EPO Publication No. 116,201 discloses the first successful application of the &agr;-factor leader to direct the expression and secretion of an heterologous protein, epidermal growth factor, from a transformed yeast. Subsequent to this work, there have been additional reports of the expression of heterologous proteins in yeast employing the &agr;-factor leader. See, e.g., Elliott et al. (1983) Proc. Nat'l Acad. Sci. USA 80:7080-7084; Bitter et al. (1984) Proc. Nat'l Acad. Sci. USA 81:5330-5334; Smith et al. (1985) Science 229:1219-1229; EPO Publication Nos. 114,695; 123,228; 123,294; 123,544; 128,773; and 206,783. See also Brake et al. in
Protein Transport and Secretion,
p. 103 (J. M. Gething ed. 1984).
The expression systems in the above reports produce a full-length &agr;-factor leader fused to a heterologous protein. While the above work demonstrates that the &agr;-factor expression system is widely useful, it is not generally predictable prior to performing the experiment whether a particular heterologous protein will be successfully secreted, processed and biologically active. See, e.g., EPO 206,783, supra, pp. 2-5; Rothblatt et al. (1987) EMBO J. 6:3455-3463; V. L. MacKay, “Secretion of Heterologous Proteins in Yeast” (in press).
There have been several reports, based on unpublished data, that deletions from the “pro” region of the &agr;-factor leader (between the signal peptide and the first spacer) causes substantial declines in the amount of non-yeast protein secreted by yeast transformed the heterologous constructs. Sidhu et al. (1987) Gene 54:175-184, reports that yeast acid phosphatase (PHO5) is secreted into the medium from a heterologous construct employing both a full-length &agr;-factor leader, and a truncated &agr;-factor leader, but that expression levels are 3-4× less than for the PHO5 gene under the control of its homologous leader. It has also been reported that deletions in the prepro-&agr;-factor precursor gene results in substantial declines in secretion of the native &agr;-factor peptide. See, e.g., V. L. MacKay, supra; Rothblatt et al., supra.
A need exists, therefore, to improve the &agr;-factor expression system, particularly for applications to non-yeast proteins that are not efficiently produced by current &agr;-factor expression constructs.
SUMMARY OF THE INVENTION
It has been surprisingly discovered that a truncated form of the &agr;-factor leader sequence can efficiently direct the expression and secretion of heterologous polypeptides in yeast. Particularly surprising is the discovery that truncated &agr;-factor leader sequences can substantially improve the efficiency of expression of such heterologous proteins in relation to expression systems using the full-length &agr;-factor leader; i.e., higher levels of correct N-terminal processing, secretion of heterologous proteins wherein a greater percentage of the molecules are biologically active, etc. These results are particularly surprising in view of reports that deletions from the leader sequence of the &agr;-factor precursor result in decreased levels of secretion of active &agr;-factor.
The present invention provides, therefore, for alternative &agr;-factor-based expression constructs, which are particularly useful for the expression of heterologous polypeptides which are either inefficiently expressed by full-length &agr;-factor leader constructs, or are not expressed at all by such full-length constructs.
In one embodiment, the present invention is directed to a yeast cell comprising a DNA construct that provides for the expression and secretion of a non-yeast protein, said DNA construct comprising a coding sequence under the control of yeast-recognized transcription initiation and termination sequences, said coding sequence encoding a precursor polypeptide comprised of a leader sequence and said non-yeast protein linked by a processing site that provides for the cleavage of said non-yeast protein from said precursor polypeptide, wherein said leader sequence is about 25 to about 50 N-terminal residues of said precursor polypeptide and comprises the signal peptide of a yeast &agr;-factor precursor and a single glycosylation site.
In another embodiment, the present invention is directed to a double-stranded DNA molecule comprising a region encoding a precursor polypeptide secretable by a yeast host, said region, with reference to one of the DNA strands, having the structure:
5′-AF-CHO-X
n
-S-Gene*-3′
wherein
AF encodes a yeast &agr;-factor signal peptide; CHO encodes a glycosylation site;
X
n
encodes a polypeptide of n amino acids in length that does not contain a glycosylation site or a processing site that provides for cleavage of said precursor polypeptide in vivo by yeast;
n is an integer from 0 to about 30;
Gene* encodes a non-yeast protein; and
S encodes a processing site that provides for cleavage of said precursor polypeptide.
The present invention is also directed to methods of employing the above cells and DNA constructs to produce recombinant proteins, as well as the compositions of recombinant proteins produced by the above methods. Other embodiments will also be readily apparent to those of ordinary skill in the art.
REFERENCES:
patent: 4546082 (1985-10-01), Kurjan et al.
patent: 4870008 (1989-09-01), Brake
patent: 5302697 (1994-04-01), Goodey et al.
patent: 088632 (1983-09-01), None
patent: 0 123 228 (1984-10-01), None
patent: 0 123 294 (1984-10-01), None
patent: 0 171 000 (1986-12-01), None
patent: 0 206 783 (1986-12-01), None
patent: 0 214 826 (1987-03-01), None
Barr, P.J. et al. (eds.),Yeast Genetic Engineering, 1989, 239 and 272.
Brake et al.,Mol. Cell. Biol., 1983, 3(8), 1440-1450.
Fuller et al.,Microbiology, 1986, 273-278.
Hitzeman et al.,Science, 1983, 219, 620-625.
Julius et al.,Cell, 1983, 32, 839-852.
Rothblatt et al.,EMBO J., 1987, 6(11), 3455-3463.
Singh et al., “Saccharomyces cerevisiaecontains two discrete genes coding for the &agr;-factor pheromone,”Nucl. Acids Res., 1983, 11(12), 4049-4063.
Sidhu et al.,Gene, 1987, 54, 175-184.
Zsebo, K.M. et al.,J. Biol. Chem., 1986, 261(13), 5858-5865.
Blackburn Robert P.
Chiron Corporation
Guth Joseph H.
Paintin Francis A.
Prouty Rebecca E.
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