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US 6,183,985 B2Granted
High level expression of proteins in yeast
AssigneeChiron Corporation
InventorJeffrey R. Shuster
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Abstract
A method is provided for expressing non-yeast proteins under the control of ADH2 regulatory sequences in a yeast host having enhanced expression of ADR I. Transformed yeast hosts are also provided.
Claims10
A transformed yeast host comprising:
- (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 an ADH2/GAPD promoter and 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, said transcription initiation sequence comprising a GAPD promoter.
A method of expressing a non-yeast protein in a yeast host comprising:
- (A) providing a yeast host that comprises:
- (i) a first expression cassette comprising (a) a first DNA coding sequence that encodes a non-yeast protein, (b) a first yeast-recognized transcription initiation sequence which is a GAPD promoter, and (c) a first yeast-recognized termination sequence,
- wherein the first DNA coding sequence is under the control of the first transcription initiation sequence and the first termination sequence, and
- wherein the first transcription initiation sequence comprises a yeast ADH2 upstream activation site and a first yeast promoter heterologous to the ADH2 upstream activation site;
- (ii) a second expression cassette heterologous to the yeast host comprising (a) second DNA coding sequence that encodes ADR I, (b) a second yeast-recognized transcription initiation sequence, and (c) a second yeast-recognized termination sequence,
- wherein the second DNA coding sequence is under the control of the second transcription initiation sequence and the second termination sequence, and
- wherein the second transcription initiation sequence comprises a second yeast promoter;
- (B) culturing a clonal population of the yeast host under condition whereby the non-yeast protein and ADR I are expressed;
- and
- (C) recovering the non-yeast protein from the clonal population.
7 more elements
A transformed yeast host comprising:
- (i) a first expression cassette comprising (a) a first DNA coding sequence that encodes a non-yeast protein, (b) a first yeast-recognized transcription initiation sequence, and (c) a first yeast-recognized termination sequence,
- wherein the first DNA coding sequence is under the control of the first transcription initiation sequence and the first termination sequence, and
- wherein the first transcription initiation sequence comprises a yeast ADH2 upstream activation site and a first yeast promotor heterologous to the ADH2 upstream activation site;
- (ii) a second expression cassette heterologous to the yeast host comprising (a) a second DNA coding sequence that encodes ADR I;
- (b) a second yeast-recognized transcription initiation sequence, and (c) a second yeast-recognized termination sequence,
- wherein the second DNA coding sequence is under the control of the second transcription initiation sequence and the second termination sequence,
- wherein the second transcription initiation sequence comprises a second yeast promotor, and
- wherein said second expression cassette is integrated into the genome of the yeast.
5 more elements
Dependencies are inferred from the claim text.