Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1995-03-01
2001-02-06
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S254210
Reexamination Certificate
active
06183985
ABSTRACT:
TECHNICAL FIELD
The present invention is directed to methods and materials useful for the production of heterologous proteins in yeast by recombinant DNA methods. More particularly, the present invention is directed to methods and materials which improve the yield of heterologous proteins made in yeast when said yeast are expressed under the control of a yeast alcohol dehydrogenase isoenzyme II (ADH II) gene promoter or its upstream activation sequence (UAS).
BACKGROUND
The expression of foreign genes in yeast regulated by the system which causes the expression of ADH II has proved to be quite valuable. Such expression usually involves placing the sequence encoding the heterologous protein under the control of the promoter from the ADH2 gene, or hybrid promoters employing the upstream regulatory region of the ADH2 promoter in combination with a strong yeast promoter, such as the glyceraldehyde-3-phosphate (GAP) promoter. See, e.g, EPO Pub. No. 164,556; EPO Pub. No. 196,056; commonly owned U.S. patent application Ser. No. 868,639, filed May 29, 1986. Expression cassettes for the heterologous protein employing the ADH2 regulatory regions are usually present in the yeast expression host in high copy numbers. While good results are obtained with such expression systems, a continuing need exists to enhance heterologous protein yield.
A number of studies have been published regarding regulation of the ADH2 gene by the yeast ADR1 gene. See, e.g., Shuster et al. (1986) Mol. Cell. Biol. 6:1894-1902; Denis et al. (1983) Mol. Cell. Biol. 3:360-370. Several studies have been published examining the relationship between the expression level of ADR1 and the expression level of native ADH2 genes.
See Irani et al. (1987) Mol. Cell. Biol. 7:1233-1241; Denis (1987) Mol. Gen. Genet. 208:101-106. These studies however, did not demonstrate whether increased ADR1 expression would ultimately improve the yield of a heterologous protein expressed under the control of ADH2 regulatory sequences. For example, it was observed by Irani et al. (1987) that over one hundred copies of ADR1 did not overcome the 3- to 4-fold inhibition in ADH2 transcription caused by multiple ADH2 promoters. It was suggested that this result supports a model of ADH II regulation in which there are positive limiting factors on ADH2 expression other than ADR1. Denis (1987) also reported that increasing ADR1 gene dosage was apparently toxic to the yeast host, resulting in a significant increase in cell doubling time.
SUMMARY OF THE INVENTION
It has been surprisingly discovered that the yield of heterologous protein by a yeast host transformed with an expression cassette for the heterologous protein employing the ADH2 regulatory system can be significantly enhanced by increasing the expression in the yeast host of the protein encoded by the ADR1 gene. This result is surprising in view of the reported toxicity of increased ADR1 gene dosage and the minimal effect of 100+ copies of the ADR1 gene on ADH II activity in a yeast host transformed with multiple ADH2 promoters.
In one embodiment, the present invention is directed to a method of expressing a heterologous protein in yeast comprising: providing a yeast host transformed by: (i) an expression cassette for said non-yeast protein comprising a DNA coding sequence for said non-yeast protein under the control of yeast-recognized transcription initiation and termination sequences functional in said yeast host, said transcription initiation sequence further comprising a yeast ADH2 upstream activation site; and (ii) an expression cassette for yeast ADR2 comprising a DNA coding sequence for said ADR I under the control of yeast-recognized transcription initiation and termination sequences functional in said yeast host; (b) culturing a clonal population of said transformed yeast host under conditions whereby said non-yeast protein and said ADR I are expressed; and (c) recovering said non-yeast protein from said clonal population.
In another embodiment, the present invention is directed to a yeast cell transformed by: (a) an expression cassette comprising a DNA coding sequence for a non-yeast protein under the control of yeast-recognized transcription initiation and termination sequences functional in said yeast host, said transcription initiation sequence comprising a yeast ADH2 upstream activation site; and (b) an expression cassette for ADR I comprising a DNA coding sequence for said ADR I under the control of yeast-recognized transcription initiation and termination sequences functional in said yeast host.
In yet another embodiment, the present invention comprises a transformed yeast comprising an expression cassette integrated into the genome containing a coding sequence for ADR I under the control of yeast-recognized transcription initiation and termination sequences, said transcription initiation sequence being other than the ADR1 promoter.
These and other embodiments of the present invention will be readily apparent to those of ordinary skill in the art from the following description.
REFERENCES:
patent: 4880734 (1989-11-01), Burke et al.
patent: 0196056 (1986-10-01), None
patent: 0234871 (1987-09-01), None
patent: 0252737 (1988-01-01), None
Hartshorne et al.,Nature(1986) 320:283-287.
Bemis et al., (1988) Molec. and Cellular Biol. 8(5) :2125-2131.
Blumberg et al., (1988) Molec. and Cellular Biol. 8(5) :1868-1876.
Denis, (1987) Mol. Gen. Genet. 208:101-106.
Irani et al., (1987) Molec. and Cellular Biol. 7(3) :1233-1241.
Schultz et al., (1987) Gene 61:123-133.
Blackburn Robert P.
Chiron Corporation
Ketter James
Morley Kimberlin L.
Potter Jane E. R.
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