US4588684A
Expired / Lapsed
US
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a-Factor and its processing signals
Assignee: Novartis Vaccines and Diagnostics Inc
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Filing: 1983-04-26
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Granted: 1986-05-13
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Expires: 2003-04-26
Abstract
Abstract Novel DNA constructs are provided for efficient expression of polypeptides by yeasts. The constructs employ yeast a-factor secretion leader and processing signals joined to a DNA sequence encoding a polypeptide of interest in reading frame with the a-factor signals. The constructions provide for the expression, secretion and maturation of the desired polypeptide. A strategy is provided for the isolation of the a-factor secretion leader and processing signals and the joining, by means of a relatively short adaptor, molecules of the DNA sequence encoding the polypeptide to the processing signals in proper reading frame. The bacterial cell strain E. coli HB101 (pAB163) was deposited at the A.T.C.C. on Apr. 20, 1983 and given Accession No. 39342.
Inventors
- 👤 Anthony J. Brake
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-01 11:38
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C07; C07K; C07K14/00; C07K14/37; C07K14/39; C12N15/63; C12N15/79; C12N15/80; C12N15/81; C07K2319/00; C07K2319/01; C07K2319/02; Y; Y10; Y10S; Y10S930/00; Y10S930/01; Y10S930/30
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
27 words
A DNA construct comprising the yeast leader sequence of a-factor including processing signals and a heterologous gene in reading frame with said leader sequence and processing signals.
Claim 2
Dependent Claim
27 words
A DNA construct comprising the yeast leader sequence of a-factor including processing signals and a heterologous gene in reading frame with said leader sequence and processing signals.
Claim 3
Dependent Claim
38 words
A DNA construct according to claim 1 of the formula: L-(PS-(a-factor)).sub.n -PS-gene wherein: L is the a-factor secretory leader sequence; PS is the processing signal; gene is a gene heterologous to yeast; and n is 0 or 1.
Claim 4
Dependent Claim
17 words
A DNA construct according to claim 2, wherein said gene is a mammalian gene or portion thereof.
Claim 5
Dependent Claim
18 words
A DNA construct according to claim 2, wherein said gene is a mammalian pathogen gene or portion thereof.
Claim 6
Dependent Claim
107 words
A DNA construct of the formula: P-L-(PS-(a-factor)).sub.n -(PS-(gene).sub.r).sub.w -|RepS|.sub.q wherein: P is a promoter recognized by yeast RNA polymerase; L is the a-factor secretory leader sequence; PS is the processing signal; gene is a DNA sequence having an open reading frame in phase with L and PS encoding for a polypeptide heterologous to yeast; q and w are at least one; |RepS| is a replication system recognized by yeast and may be located anywhere in the construct external to the immediate expression region defined by P and gene as extremities; and n and r are 0 or 1, at least one of n or r being 1.
Claim 7
Dependent Claim
107 words
A DNA construct of the formula: P-L-(PS-(a-factor)).sub.n -(PS-(gene).sub.r).sub.w -|RepS|.sub.q wherein: P is a promoter recognized by yeast RNA polymerase; L is the a-factor secretory leader sequence; PS is the processing signal; gene is a DNA sequence having an open reading frame in phase with L and PS encoding for a polypeptide heterologous to yeast; q and w are at least one; |RepS| is a replication system recognized by yeast and may be located anywhere in the construct external to the immediate expression region defined by P and gene as extremities; and n and r are 0 or 1, at least one of n or r being 1.
Claim 8
Dependent Claim
17 words
A construct according to claim 5, wherein r is 1 and said polypeptide is a mammalian polypeptide.
Claim 9
Dependent Claim
21 words
A construct according to claim 5, wherein r is 1 and said polypeptide is a mammalian pathogen gene or portion thereof.
Claim 10
Dependent Claim
12 words
A construct according to claim 5, wherein said promoter is a-factor promoter.
Claim 11
Dependent Claim
22 words
A construct aqcording to claim 5 wherein said replication system recognized by yeast is the yeast 2 μm plasmid or portion thereof.
Claim 12
Dependent Claim
19 words
A construct according to claim 5 wherein q is 2 and further comprising a replication system recognized by bacteria.
Claim 13
Independent Claim
149 words
A method for producing a secreted polypeptide product which comprises: growing yeast cells containing a DNA construct of the formula: P-L-(PS-(a-factor)).sub.n -(PS-(gene).sub.r).sub.w -|RepS|.sub.q wherein: P is a promoter recognized by yeast RNA polymerase; L is the a-factor secretory leader sequence; PS is the processing signal; gene is a DNA sequence having an open reading frame in phase with L and PS encoding for a polypeptide heterologous to yeast; q and w are at least one; |RepS| is a replication system recognized by yeast and may be located anywhere in the construct external to the immediate expression region defined by P and gene as extremities; and n and r are 0 or 1, at least one of n or r being 1; whereby said secretory leader sequence, processing signals and gene encoding for said polypeptide are expressed as a fused polypeptide, which is secreted and processed by said yeast cells.
Claim 14
Independent Claim
149 words
A method for producing a secreted polypeptide product which comprises: growing yeast cells containing a DNA construct of the formula: P-L-(PS-(a-factor)).sub.n -(PS-(gene).sub.r).sub.w -|RepS|.sub.q wherein: P is a promoter recognized by yeast RNA polymerase; L is the a-factor secretory leader sequence; PS is the processing signal; gene is a DNA sequence having an open reading frame in phase with L and PS encoding for a polypeptide heterologous to yeast; q and w are at least one; |RepS| is a replication system recognized by yeast and may be located anywhere in the construct external to the immediate expression region defined by P and gene as extremities; and n and r are 0 or 1, at least one of n or r being 1; whereby said secretory leader sequence, processing signals and gene encoding for said polypeptide are expressed as a fused polypeptide, which is secreted and processed by said yeast cells.
References Cited (Backward Citations)
US4338397A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1989-05-12 | 1989-11-11 | 1990-05-13 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-05-12 | 1993-11-11 | 1994-05-13 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-05-12 | 1997-11-11 | 1998-05-13 | $7700.00 | Window Closed / Lapsed |