US4994379A
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Modified signal peptides
Assignee: Novartis Vaccines and Diagnostics Inc
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Filing: 1989-08-22
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Granted: 1991-02-19
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Expires: 2009-08-22
Abstract
Abstract The invention discloses modified signal peptides derived from wild-type signal peptides of the type that are capable of forming membrane-bound lipoproteins and methods for making such modified signal peptides and DNA sequences encoding them. Modified signal peptides of the invention and DNA sequences encoding them are useful for increasing the secretion of heterologous gene products produced by transformed host organisms. The invention further discloses a method for producing recombinant DNA sequences in vivo.
Inventors
- 👤 Shing Chang
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 06:08
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C07K14/54; C07K14/55; C07K14/575; C07K14/61; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/11; C12N15/62; C12N15/625; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/034; C07K2319/036; C07K2319/70; C07K2319/74; C07K2319/75
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
153 words
A DNA sequence encoding a modified exo-signal peptide, said modified exo-signal peptide corresponding to a wild-type signal peptide which a) is capable of forming membrane bound lipoproteins and b) includes a facilitating region within the carboxy-terminal region thereof, wherein said wild-type signal peptide is a bacterial signal peptide selected from the group consisting of B licheniformis penicillinase signal sequence, E. coli lipoprotein signal sequence. Bacillus cereus penicillinase III signal sequence, and S. aureus penicillinase signal sequence said facilitating region comprising a tetrapeptide, selected from the group consisting of leu-ala-gly-cys and leu-ser-ala-cys, said facilitating region having a single cysteine, the presence of which is necessary for the formation of membrane bound lipoprotein, wherein the modification to said exo-signal peptide comprises another amino acid X in place in said cysteine of the facilitating region, said amino acid X not forming a membrane bound lipoprotein but forming substantially more exoprotein than said wild-type signal peptide sequence.
Claim 2
Dependent Claim
153 words
A DNA sequence encoding a modified exo-signal peptide, said modified exo-signal peptide corresponding to a wild-type signal peptide which a) is capable of forming membrane bound lipoproteins and b) includes a facilitating region within the carboxy-terminal region thereof, wherein said wild-type signal peptide is a bacterial signal peptide selected from the group consisting of B licheniformis penicillinase signal sequence, E. coli lipoprotein signal sequence. Bacillus cereus penicillinase III signal sequence, and S. aureus penicillinase signal sequence said facilitating region comprising a tetrapeptide, selected from the group consisting of leu-ala-gly-cys and leu-ser-ala-cys, said facilitating region having a single cysteine, the presence of which is necessary for the formation of membrane bound lipoprotein, wherein the modification to said exo-signal peptide comprises another amino acid X in place in said cysteine of the facilitating region, said amino acid X not forming a membrane bound lipoprotein but forming substantially more exoprotein than said wild-type signal peptide sequence.
Claim 3
Dependent Claim
10 words
The DNA sequence of claim 1 wherein X is serine.
Claim 4
Dependent Claim
15 words
The DNA sequence of claim 1 fused to a DNA sequence encoding a desired protein.
Claim 5
Dependent Claim
17 words
The DNA sequence of claim 3 wherein the desired protein is a bacterial exopenicillinase, IL-2, or hGH.
Claim 6
Dependent Claim
17 words
A replicable expression vector effective in procaryotic host cells to express the DNA sequence of claim 3.
Claim 7
Dependent Claim
21 words
An expression vector replicable in procaryotic host cells comprising the DNA sequence of claim 3 operably linked to suitable control sequences.
Claim 8
Dependent Claim
10 words
Recombinant host cells transformed with the vector of claim 6.
Claim 9
Dependent Claim
178 words
A replicable expression vector capable in prokaryotic cells of effecting the production of a peptide and secretion of an exoprotein comprising a modified exosignal peptide operably linked to a desired protein, said modified exo-signal peptide corresponding to a wild-type signal peptide which is a) capable of forming membrane bound lipoproteins and b) includes a facilitating region within the carboxy terminal region thereof, wherein said wild-type signal peptide is a bacterial signal peptide selected from the group consisting of B licheniformis penicillinase signal sequence, E. coli lipoprotein signal sequence. Bacillus cereus penicillinase III signal sequence, and S. aureus penicillinase signal sequence, said facilitating region comprising a tetrapeptide selected from the group consisting of leu-ala-gly-cys and leu-ser-ala-cys, said facilitating region having a single cysteine, the presence of which is necessary for the formation of a membrane bound lipoprotein wherein the modification to said exo-signal peptide comprises another amino acid X in place of said cysteine of the facilitating region, said amino acid X not forming a membrane bound lipoprotein but forming substantially more exoprotein than said wildtype signal peptide sequence.
Claim 10
Dependent Claim
178 words
A replicable expression vector capable in prokaryotic cells of effecting the production of a peptide and secretion of an exoprotein comprising a modified exosignal peptide operably linked to a desired protein, said modified exo-signal peptide corresponding to a wild-type signal peptide which is a) capable of forming membrane bound lipoproteins and b) includes a facilitating region within the carboxy terminal region thereof, wherein said wild-type signal peptide is a bacterial signal peptide selected from the group consisting of B licheniformis penicillinase signal sequence, E. coli lipoprotein signal sequence. Bacillus cereus penicillinase III signal sequence, and S. aureus penicillinase signal sequence, said facilitating region comprising a tetrapeptide selected from the group consisting of leu-ala-gly-cys and leu-ser-ala-cys, said facilitating region having a single cysteine, the presence of which is necessary for the formation of a membrane bound lipoprotein wherein the modification to said exo-signal peptide comprises another amino acid X in place of said cysteine of the facilitating region, said amino acid X not forming a membrane bound lipoprotein but forming substantially more exoprotein than said wildtype signal peptide sequence.
Claim 11
Dependent Claim
10 words
Recombinant host cells transformed with the vector of claim 8.
Claim 12
Dependent Claim
24 words
A method of effecting secretion of a desired protein sequence which method comprises culturing the transformed cells of claim 9 in suitable nutrient medium.
Claim 13
Independent Claim
195 words
A method of obtaining secretion of a desired protein, which method comprises transforming prokaryotic host cells with a replicable expression vector capable in prokaryotic cells of effecting the production of a peptide and secretion of an exoprotein comprising a modified exo-signal peptide operably linked to a desired protein, said modified exo-signal peptide operably linked to a desired protein which is a) capable of forming membrane bound lipoproteins and b) includes a facilitating region within the carboxy terminal region thereof, wherein said wildtype signal peptide is a bacterial signal peptide selected from the group consisting of B licheniformis penicillinase signal sequence, E. coli lipoprotein signal sequence, Bacillus cereus penicillinase III signal sequence, and S. aureus penicillinase signal sequence, said facilitating region comprising a tetrapeptide selected from the group consisting of leu-ala-gly-cys and leu-serala-cys, said facilitating region having a single cysteine, the presence of which is necessary for the formation of a membrane bound lipoprotein wherein the modification to said exo-signal peptide comprises another amino acid X in place of said cysteine of the facilitating region, said amino acid X not forming a membrane bound lipoprotein but forming substantially more exoprotein that said wild-type signal peptide sequence.
Claim 14
Independent Claim
195 words
A method of obtaining secretion of a desired protein, which method comprises transforming prokaryotic host cells with a replicable expression vector capable in prokaryotic cells of effecting the production of a peptide and secretion of an exoprotein comprising a modified exo-signal peptide operably linked to a desired protein, said modified exo-signal peptide operably linked to a desired protein which is a) capable of forming membrane bound lipoproteins and b) includes a facilitating region within the carboxy terminal region thereof, wherein said wildtype signal peptide is a bacterial signal peptide selected from the group consisting of B licheniformis penicillinase signal sequence, E. coli lipoprotein signal sequence, Bacillus cereus penicillinase III signal sequence, and S. aureus penicillinase signal sequence, said facilitating region comprising a tetrapeptide selected from the group consisting of leu-ala-gly-cys and leu-serala-cys, said facilitating region having a single cysteine, the presence of which is necessary for the formation of a membrane bound lipoprotein wherein the modification to said exo-signal peptide comprises another amino acid X in place of said cysteine of the facilitating region, said amino acid X not forming a membrane bound lipoprotein but forming substantially more exoprotein that said wild-type signal peptide sequence.
Claim 15
Dependent Claim
44 words
A replicable expression vector effective in pyrocaryotic recombinant host cells in expressing a DNA sequence encoding an exo-signal peptide which comprises an amino acid sequence Met-Lys-Leu-Trp-Phe-Ser-Thr-Leu-Lys-Leu-Lys-Lys-Ala-Ala-Ala-Val-Leu-LeuPhe-Ser-Cys-Val-Ala-Leu-Ala-Gly-X-Y-(Asn-Asn-Gln-Thr-Asn-Ala) n wherein X is serine or alanine, Y is alanine or proline, and n is 0 or 1.
Claim 16
Dependent Claim
44 words
A replicable expression vector effective in pyrocaryotic recombinant host cells in expressing a DNA sequence encoding an exo-signal peptide which comprises an amino acid sequence Met-Lys-Leu-Trp-Phe-Ser-Thr-Leu-Lys-Leu-Lys-Lys-Ala-Ala-Ala-Val-Leu-LeuPhe-Ser-Cys-Val-Ala-Leu-Ala-Gly-X-Y-(Asn-Asn-Gln-Thr-Asn-Ala) n wherein X is serine or alanine, Y is alanine or proline, and n is 0 or 1.
Claim 17
Dependent Claim
10 words
Recombinant host cells transformed with the vector of claim 12.
References Cited (Backward Citations)
No citation records indexed.
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| 3.5 Years (Year 4) | 1994-02-18 | 1994-08-20 | 1995-02-19 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1998-02-18 | 1998-08-20 | 1999-02-19 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2002-02-18 | 2002-08-20 | 2003-02-19 | $7700.00 | Window Closed / Lapsed |