EP0179449A1
Expired / Lapsed
EP
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Streptomyces secretion vector
Assignee: Biotechnica International Inc
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Filing: 1985-10-22
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Granted: —
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Expires: 2005-10-22
Abstract
Abstract A host Streptomyces cell engineered to produce and secrete a desired protein by the introduction of a DNA vehicle that does not occur naturally in the cell. The vehicle comprises: 1) signal-sequence-encoding DNA derived from a Streptomyces endoH gene; 2) a structural gene encoding the protein; and 3) regulatory DNA that includes a promoter sequence positioned to be effective to begin transcription of a) and b) in the host cell, as well as DNA encoding a ribosome-binding site. Methods of using the cell to produce the desired protein are also disclosed.
Inventors
- 👤 Sonia Guterman
- 👤 Janice Pero
- 👤 Phillips Robbins
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-05 07:32
CPC Classifications
C; C12; C12Y; C12Y302/00; C12Y302/01; C12Y302/01096; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/74; C12N15/76; C12N9/00; C12N9/14; C12N9/16; C12Y301/00; C12Y301/03; C12Y301/03001; C07; C07K; C07K2319/00; C07K2319/01; C07K2319/02; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/886; Y10S435/896; Y10S930/00; Y10S930/01; Y10S930/30
Claims Analysis
Total Claims: 35
Claim 1
Independent Claim
69 words
A recombinant expression vector comprising a) a signal-sequence-encoding DNA derived from a Streptomyces endo H gene; b) optionally a structural gene encoding a desired protein inserted in a suitable restriction site; and c) a regulatory DNA comprising a promoter sequence and a DNA which encodes a ribosome-binding site, the DNA of a) and b) being transcribed and translated in a.Streptomyces host cell under the control of said regulatory DNA.
Claim 2
Dependent Claim
69 words
A recombinant expression vector comprising a) a signal-sequence-encoding DNA derived from a Streptomyces endo H gene; b) optionally a structural gene encoding a desired protein inserted in a suitable restriction site; and c) a regulatory DNA comprising a promoter sequence and a DNA which encodes a ribosome-binding site, the DNA of a) and b) being transcribed and translated in a.Streptomyces host cell under the control of said regulatory DNA.
Claim 3
Dependent Claim
20 words
The recombinant expression vector of claim 1,characterized in that the endo H gene is derived from the species S. plicatus.
Claim 4
Dependent Claim
26 words
The recombinant expression vector of claim 1,characterized in that the promoter sequence is derived from the promoter sequence of an endo H gene.of a Streptomyces species.
Claim 5
Dependent Claim
26 words
The recombinant expression vector of claim 3,characterized in that the promoter sequence is derived from the promoter sequence of the endo H gene of S. plicatus.
Claim 6
Dependent Claim
25 words
The recombinant expression vector of claim 1,characterized in that the promoter sequence is derived from the promoter sequence of the aph gene of S. fradiae.
Claim 7
Dependent Claim
29 words
The recombinant expression vector of claim 1,characterized in that the ribosome-binding site DNA is derived from the ribosome-binding site DNA of an endo H gene of a Streptomyces species.
Claim 8
Dependent Claim
28 words
The recombinant expression vector of claim 1, characterized in that the regulatory DNA is derived from the regulatory DNA of the endo H gene of a Streptomyces species.
Claim 9
Dependent Claim
37 words
The recombinant expression vector of claim 2, characterized in that the promoter sequence and the ribosome-binding site DNA is derived from the respective promoter sequence and ribosome-binding site DNA of the endo H gene of S. plicatus.
Claim 10
Dependent Claim
45 words
The recombinant expression vector of claim 8, characterized in that it comprises the approximately 1.6 kb fragment of plasmid pEHB1.6 between the EcoRI and BamHI restriction sites, said fragment comprising the promoter, ribosome-binding site and structural gene of the endo H gene of S. plicatus.
Claim 11
Dependent Claim
58 words
The recombinant expression vector of claim 1, characterized in that the signal sequence encoding DNA comprises DNA that codes for the following amino acid sequence: Met Phe Thr Pro V al A rg A rg Arg Val Arg Thr Ala Ala Leu Ala Leu Ser Ala Ala Ala Ala Leu Val Leu Gly Ser Thr Ala Ala Ser.
Claim 12
Dependent Claim
19 words
The recombinant expression vector of claim 1,charac- terized in that the signal sequence encoding DNA comprises the following sequence:
Claim 13
Dependent Claim
66 words
The recombinant expression vector of claim 11,characterized in that the signal sequence encoding DNA comprises DNA that codes for the following amino acid sequence: Met Phe Thr Pro Val Arg Arg Arg Val Arg Thr Ala Ala Leu Ala Leu Ser Ala Ala Ala Ala Leu Val Leu Gly Ser Thr Ala Ala Ser Gly Ala Ser Ala Thr Pro Ser Pro Ala Pro Ala Pro.
Claim 14
Dependent Claim
19 words
The recombinant expression vector of claim 12, characterized in that the signal sequence encoding DNA comprises the following sequence:
Claim 15
Dependent Claim
23 words
The recombinant expression vector of claim 1, characterized in that it comprises at least one replication sequence derived from the Streptomyces plasmid pIJ702.
Claim 16
Dependent Claim
21 words
The recombinant expression vector of claim 1, characterized in that it comprises at least one sequence enabling replication in E. coli.
Claim 17
Dependent Claim
21 words
The recombinant expression vector of claim 1,characterized in that it comprises a DNA sequence capable of conferring antibiotic resistance on Streptomyces.
Claim 18
Dependent Claim
16 words
The recombinant expression vector of claim 16,characterized in that said DNA sequence confers resistance to the-thiostrepton.
Claim 19
Dependent Claim
24 words
The recombinant expression vector of claim 15,characterized in that it also includes a DNA, sequence capable of conferring antibiotic resistance on E. coli cells.
Claim 20
Dependent Claim
20 words
The recombinant expression vector of claim 18,characterized in that antibiotic-resistance and replication- enabling sequences are derived from the plasmid pBR322.
Claim 21
Dependent Claim
17 words
The recombinant expression vector of claim 1, characterized in that the structured gene codes for endo H.
Claim 22
Dependent Claim
26 words
The recombinant expression vector of claim 20,characterized in that it comprises an intact endo H gene, including a signal sequence encoding DNA and a regulatory DNA.
Claim 23
Dependent Claim
19 words
The recombinant expression vector of claim 21,characterized in that the intact endo H gene is derived from S. plicatus.
Claim 24
Dependent Claim
26 words
The recombinant expression vector of claim 1, characterized in that the structural gene is one which is not naturally transcribed and translated with said signal-sequence-encoding DNA.
Claim 25
Dependent Claim
26 words
The recombinant expression vector of claim 23,characterized in that the structural gene is derived from a gene which naturally occurs in a genus other than Streptomyces.
Claim 26
Dependent Claim
21 words
The recombinant expression vector of claim 24,characterized in that the structural gene is derived from an E. coli alkaline phosphatase gene.
Claim 27
Dependent Claim
19 words
The recombinant expression vector of claim 1, characterized in that the restriction site is Sph I or Hind III.
Claim 28
Dependent Claim
19 words
The recombinant expression vector pGH202 (ATCC 39896), pGH205 (ATCC 39895), or a recombinant expression vector derived from these vectors.
Claim 29
Dependent Claim
19 words
The recombinant expression vector pGH202 (ATCC 39896), pGH205 (ATCC 39895), or a recombinant expression vector derived from these vectors.
Claim 30
Dependent Claim
56 words
A host cell transformed with a recombinant expression vector according to any one of claims 1 - 27 belonging to the species S.lividans, S. coelicolor, S. fradiae, S. griseofuscus, S. reticuli, S. rimosus, S. albus, S. parvulus, S. ambofaciens, S. aureofaciens, S. plicatus, S. espinosus, S. lincolnensis, S. erythraeus, S. antibioticus, S. griseus, or S. glaucens.
Claim 31
Dependent Claim
16 words
The host cell according to claim 28,characterized in that it is S. lividans or S. coelicolor.
Claim 32
Dependent Claim
42 words
A host cell of a first Streptomyces species transformed with a recombinant expression vector according to claim 1 containing an endo H signal sequence DNA which is derived from an endo H gene of a second species, different from said first species.
Claim 33
Dependent Claim
31 words
The host cell according to claim 30,characterized in that it belongs to the species S. lividans and in that the recombinant expression vector contains the endo H gene of S. plicatus.,
Claim 34
Dependent Claim
39 words
Use of a recombinant expression vector according to any one of claims 1 - 27 for the expression of a structural gene encoding a desired protein in a host organism according to any one of claims 28 - 31.
Claim 35
Independent Claim
32 words
A method of producing a desired protein comprising culturing a Streptomyces cell according to any one of claims 28 - 31 in a culture medium and recovering said protein from the medium.
References Cited (Backward Citations)
EP0148552A1
Cited
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