US5716808A
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Genetic engineering of pichia methanolica
Assignee: Zymogenetics Inc
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Filing: 1996-08-26
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Granted: 1998-02-10
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Expires: 2016-08-26
Abstract
Abstract Methods for preparing Pichia methanolica cells containing foreign DNA constructs and methods for producing foreign polypeptides in Pichia methanolica cells are disclosed. The cells are transformed with a DNA construct comprising a first DNA segment comprising a transcription promoter of a methanol-inducible P. methanolica gene, a second DNA segment encoding a higher eukaryotic polypeptide, a third DNA segment comprising a P. methanolica gene transcription terminator, and a selectable marker. For production of foreign polypeptides, the cells are cultured under conditions in which the second DNA segment is expressed, and the polypeptide encoded by the second DNA segment is recovered.
Inventors
- 👤 Christopher K. Raymond
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 22:15
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12N9/0065; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/80; C12N15/81; C12N15/815; C12N9/0006; C12N9/14; C12N9/48; C12N9/50; C12N9/58; C12N9/60; C12N9/88
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
96 words
A method for producing a higher eukaryotic polypeptide in Pichia methanolica comprising: (a) culturing a P. methanolica cell into which has been introduced a heterologous DNA construct comprising the following operably linked elements: (i) a first DNA segment comprising a transcription promoter of a methanol-inducible P. methanolica gene; (ii) a second DNA segment encoding a higher eukaryotic polypeptide; (iii) a third DNA segment comprising a P. methanolica gene transcription terminator; and (iv) a selectable marker, under conditions in which the second DNA segment is expressed; and (b) recovering the polypeptide encoded by the second DNA segment.
Claim 2
Independent Claim
96 words
A method for producing a higher eukaryotic polypeptide in Pichia methanolica comprising: (a) culturing a P. methanolica cell into which has been introduced a heterologous DNA construct comprising the following operably linked elements: (i) a first DNA segment comprising a transcription promoter of a methanol-inducible P. methanolica gene; (ii) a second DNA segment encoding a higher eukaryotic polypeptide; (iii) a third DNA segment comprising a P. methanolica gene transcription terminator; and (iv) a selectable marker, under conditions in which the second DNA segment is expressed; and (b) recovering the polypeptide encoded by the second DNA segment.
Claim 3
Dependent Claim
35 words
The method according to claim 1 wherein the transcription promoter is a DNA segment that is contained within a 1.5 kilobase pair region 5' of and adjacent to a P. methanolica structural gene coding sequence.
Claim 4
Dependent Claim
34 words
The method according to claim 1 wherein the transcription terminator is a sequence that is contained within a 500 base pair region 3' of and adjacent to a P. methanolica structural gene coding sequence.
Claim 5
Dependent Claim
24 words
The method according to claim 1 wherein the culturing step is carried out in a liquid medium comprising methanol and a repressing carbon source.
Claim 6
Dependent Claim
31 words
The method according to claim 1 wherein the cells are cultured to a density of from 80 to 400 grams of wet cell paste per liter prior to recovering the polypeptide.
Claim 7
Dependent Claim
15 words
The method according to claim 1 wherein the selectable marker is a P. methanolica gene.
Claim 8
Dependent Claim
20 words
The method according to claim 1 wherein the selectable marker is a gene from a fungus other than P. methanolica.
Claim 9
Dependent Claim
15 words
The method according to claim 1 wherein the selectable marker is a dominant selectable marker.
Claim 10
Dependent Claim
13 words
The method according to claim 1 wherein the polypeptide is a human polypeptide.
Claim 11
Dependent Claim
16 words
The method according to claim 1 wherein said heterologous DNA construct is a linear DNA molecule.
Claim 12
Dependent Claim
27 words
The method according to claim 2 wherein said structural gene encodes an enzyme selected from the group consisting of alcohol oxidase, dihydroxyacetone synthase, formate dehydrogenase, and catalase.
Claim 13
Dependent Claim
13 words
The method according to claim 4 wherein said repressing carbon source is glucose.
Claim 14
Dependent Claim
16 words
The method according to claim 6 wherein the selectable marker is a P. methanolica ADE2 gene.
Claim 15
Dependent Claim
15 words
The method according to claim 7 wherein the selectable marker is a Saccharomyces cerevisiae gene.
Claim 16
Dependent Claim
28 words
The method according to claim 11 wherein the first DNA segment comprises a sequence of nucleotides as shown in SEQ ID NO:2 from nucleotide 24 to nucleotide 1354.
Claim 17
Dependent Claim
28 words
The method according to claim 11 wherein the first DNA segment comprises a sequence of nucleotides as shown in SEQ ID NO:9 from nucleotide 91 to nucleotide 169.
Claim 18
Dependent Claim
27 words
The method according to claim 13 wherein the ADE2 gene comprises a sequence of nucleotides as shown in SEQ ID NO:1 from nucleotide 407 to nucleotide 2851.
Claim 19
Independent Claim
101 words
A method for preparing Pichia methanolica cells containing a foreign DNA construct comprising: (a) introducing into P. methanolica cells a DNA construct comprising the following operably linked elements: (i) a first DNA segment comprising a transcription promoter of a methanol-inducible P. methanolica gene; (ii) a second DNA segment encoding a higher eukaryotic polypeptide; (iii) a third DNA segment comprising a P. methanolica gene transcription terminator; and (iv) a selectable marker; (b) culturing the P. methanolica cells from step (a) under conditions that select for growth of cells containing the DNA construct; and (c) recovering cells that grow under the selective conditions.
Claim 20
Independent Claim
101 words
A method for preparing Pichia methanolica cells containing a foreign DNA construct comprising: (a) introducing into P. methanolica cells a DNA construct comprising the following operably linked elements: (i) a first DNA segment comprising a transcription promoter of a methanol-inducible P. methanolica gene; (ii) a second DNA segment encoding a higher eukaryotic polypeptide; (iii) a third DNA segment comprising a P. methanolica gene transcription terminator; and (iv) a selectable marker; (b) culturing the P. methanolica cells from step (a) under conditions that select for growth of cells containing the DNA construct; and (c) recovering cells that grow under the selective conditions.
Claim 21
Dependent Claim
15 words
The method according to claim 18 wherein said selectable marker is a P. methanolica gene.
Claim 22
Dependent Claim
15 words
The method according to claim 18 wherein said selectable marker is a dominant selectable marker.
Claim 23
Dependent Claim
16 words
The method according to claim 19 wherein the selectable marker is a P. methanolica ADE2 gene.
Claim 24
Dependent Claim
27 words
The method according to claim 21 wherein the ADE2 gene comprises a sequence of nucleotides as shown in SEQ ID NO:1 from nucleotide 407 to nucleotide 2851.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 2001-02-09 | 2001-08-11 | 2002-02-10 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-02-09 | 2005-08-11 | 2006-02-10 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-02-09 | 2009-08-11 | 2010-02-10 | $7700.00 | Window Closed / Lapsed |