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US6001597A
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Compositions and methods for producing heterologous polypeptides in Pichia methanolica
Assignee: Zymogenetics Inc
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Filing: 1998-12-15
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Granted: 1999-12-14
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Expires: 2018-12-15
Abstract
Abstract Pichia methanolica cells in which an alcohol oxidase gene has been disrupted are disclosed. The cells may also be deficient in vacuolar protease activity. The cells are useful as hosts in recombinant protein production methods.
Inventors
- 👤 Christopher K. Raymond
- 👤 Erica Vanaja
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 22:20
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; C12N15/87; C12N9/0006; C12N9/14; C12N9/48; C12N9/50; C12N9/58; C12N9/60; C12N9/88
Claims Analysis
Total Claims: 19
Claim 1
Independent Claim
13 words
A Pichia mathanolica cell in which an alcohol oxidase gene has been disrupted.
Claim 2
Dependent Claim
13 words
A Pichia mathanolica cell in which an alcohol oxidase gene has been disrupted.
Claim 3
Dependent Claim
10 words
The cell of claim 1 wherein said gene is AUG1.
Claim 4
Dependent Claim
10 words
The cell of claim 1 wherein said gene is AUG2.
Claim 5
Dependent Claim
13 words
The cell of claim 1 wherein AUG1 and AUG2 genes have been disrupted.
Claim 6
Dependent Claim
14 words
The cell of claim 1 wherein said gene has been disrupted by a deletion.
Claim 7
Dependent Claim
14 words
The cell of claim 1 wherein said gene has been disrupted by an insertion.
Claim 8
Dependent Claim
19 words
The cell of claim 1 wherein the cell is functionally deficient in vacuolar proteases proteinase A and proteinase B.
Claim 9
Dependent Claim
12 words
The cell of claim 1 wherein the cell is auxotrophic for adenine.
Claim 10
Independent Claim
85 words
A method for preparing a polypeptide heterologous to Pichia methanolica, comprising: culturing a Pichia mathanolica cell in which an alcohol oxidase gene has been disrupted, wherein said cell contains a DNA construct comprising the following operably linked elements: a transcription promoter of a Pichia mathanolica gene; a DNA segment encoding a polypeptide heterologous to P. mathanolica; a transcription terminator of a P. mathanolica gene; and a selectable marker, under conditions whereby the DNA segment is expressed; and recovering the polypeptide encoded by the DNA segment.
Claim 11
Independent Claim
85 words
A method for preparing a polypeptide heterologous to Pichia methanolica, comprising: culturing a Pichia mathanolica cell in which an alcohol oxidase gene has been disrupted, wherein said cell contains a DNA construct comprising the following operably linked elements: a transcription promoter of a Pichia mathanolica gene; a DNA segment encoding a polypeptide heterologous to P. mathanolica; a transcription terminator of a P. mathanolica gene; and a selectable marker, under conditions whereby the DNA segment is expressed; and recovering the polypeptide encoded by the DNA segment.
Claim 12
Dependent Claim
12 words
The method of claim 9 wherein said alcohol oxidase gene is AUG1.
Claim 13
Dependent Claim
12 words
The method of claim 9 wherein said alcohol oxidase gene is AUG2.
Claim 14
Dependent Claim
13 words
The method of claim 9 wherein AUG1 and AUG2 genes have been disrupted.
Claim 15
Dependent Claim
16 words
The method of claim 9 wherein said alcohol oxidase gene has been disrupted by a deletion.
Claim 16
Dependent Claim
16 words
The method of claim 9 wherein said alcohol oxidase gene has been disrupted by an insertion.
Claim 17
Dependent Claim
19 words
The method of claim 9 wherein the cell is functionally deficient in vacuolar proteases proteinase A and proteinase B.
Claim 18
Dependent Claim
12 words
The method of claim 9 wherein the cell is auxotrophic for adenine.
Claim 19
Dependent Claim
18 words
The method of claim 16 wherein the DNA construct further comprises a selectable marker that complements adenine auxotrophy.
References Cited (Backward Citations)
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| 3.5 Years (Year 4) | 2002-12-13 | 2003-06-14 | 2003-12-14 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2006-12-13 | 2007-06-14 | 2007-12-14 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2010-12-13 | 2011-06-14 | 2011-12-14 | $7700.00 | Window Closed / Lapsed |