Yeast promoters for protein expression
In accordance with the invention, isolated nucleic acids, expression methods, host cells, expression vectors, and DNA constructs for producing proteins, and proteins produced using the expression methods are described. More particularly, nucleic acids isolated from Pichia pastoris wherein the nucleic acids have promoter activity are described. The invention also relates to expression methods, host cells, expression vectors, and DNA constructs, for using the Pichia pastoris promoters to produce proteins and polypeptides, and to the proteins and polypeptides produced using the expression methods.
1. An isolated nucleic acid wherein the sequence of the isolated nucleic acid comprises a sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4, or at least 95% identical to a fragment thereof, wherein the isolated nucleic acid comprises the sequence of a methanol-inducible Pichia pastoris promoter operably linked to a heterologous coding sequence, and wherein the fragment extends about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, or about 700 nucleotides upstream from the 3′ end of SEQ ID NO:2 or SEQ ID NO:4 wherein the fragment is a continuous fragment of SEQ ID NO:2 or SEQ ID NO:4 and wherein the fragment comprises a TATA box sequence to direct initiation of transcription, and wherein the isolated nucleic acid has promoter activity.
2. The isolated nucleic acid of claim 1 wherein the sequence of the isolated nucleic acid is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4, or at least 98% identical to the fragment thereof.
3. The isolated nucleic acid sequence of claim 1 wherein the sequence of the isolated nucleic acid is a sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4, or the fragment thereof.
4. The isolated nucleic acid of claim 1 wherein the heterologous coding sequence encodes a protein selected from the group consisting of a toxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine, a structural protein, an immunogenic protein, and a cell signaling protein.
5. The isolated nucleic acid of claim 4 wherein the protein is an enzyme for use in animal feed.
6. The isolated nucleic acid of claim 5 wherein the protein is selected from the group consisting of a mannanase, an amylase, a glucanase, a protease, a cellulase, and a xylanase.
7. The isolated nucleic acid of claim 5 wherein the protein is a phytase.
8. The isolated nucleic acid of claim 5 wherein the protein is a galactosidase.
9. An expression vector comprising the isolated nucleic acid of claim 1 .
10. A host cell comprising the expression vector of claim 9 .
11. A host cell comprising the isolated nucleic acid of claim 1 .
12. The host cell of claim 10 wherein the host cell is a Pichia species.
13. The host cell of claim 12 wherein the Pichia species is Pichia pastoris.
14. A DNA construct comprising the isolated nucleic acid of claim 1 .
15. A method of producing a protein, the method comprising the step of culturing in a culture medium a host cell comprising a first expression cassette comprising the isolated nucleic acid of claim 1 wherein the heterologous coding sequence encodes a protein, wherein the culturing is done under conditions permitting expression of the protein.
16. The method of claim 15 wherein the protein is selected from the group consisting of a toxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine, a structural protein, an immunogenic protein, and a cell signaling protein.
17. The method of claim 16 wherein the protein is an enzyme for use in animal feed.