Patent EP0449770A2 successfully refreshed from Google Patents.
EP0449770A2
Expired / Lapsed
EP
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Bacterial vectors
Assignee: Societe des Produits Nestle SA
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Filing: 1991-03-13
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Granted: —
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Expires: 2011-03-13
Abstract
Abstract The invention relates to the field of genetic engineering and provides novel DNA molecules which comprise multifunctional origins of replication and/or a DNA sequence coding for the most abundant protein in the supernatant of cultures of Lactococcus spec. , named Major Secretion Product (MSP), for its signal peptide and/or the promoter of the MSP gene. The novel DNA molecules are used for the production of novel shuttle vectors for cloning of DNA in at least E. coli and Lactococcus spec. or novel vectors for the expression of homologous or heterologous genes and the production of secreted gene products in gram positive bacteria such as Lactococcus spec. and Bacillus spec .
Inventors
- 👤 Bruno Dr. Suri
- 👤 Albert Dr. Schmitz
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-09 20:12
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/70; C12N15/74; C12N15/746; C07; C07K; C07K14/00; C07K14/195; C07K14/315; C07K14/81; C07K14/815; C12N15/75; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/853
Claims Analysis
Total Claims: 39
Claim 1
Independent Claim
179 words
A process for the preparation of a hybrid vector comprising a) the approximately 3.5 kbp EcoRI/SalI L. lactis insert of the plasmid pUCRS, or a functional fragment thereof, or b) a DNA sequence which hybridizes with said insert or with a functional fragment thereof, or comprises a promoter region which is naturally operatively linked to such a hybridizing DNA sequence, or c) a degenerate sequence of a DNA sequence which is covered in a) or b) and which encodes a signal peptide, or d) a derivative of a DNA molecule covered in a), b) or c), and/or e) the origin of replication of (I) the 2.5 Md plasmid of L. lactis 712, or of (II) the 5.2 Md plasmid of L. lactis 712, or of (III) a plasmid of the same incompatibility group as the 2.5 Md plasmid or the 5.2 Md plasmid of L. lactis 712, said method comprising A) culturing a host which comprises such hybrid vector and isolating such hybrid vector from said cultured host, or B) preparing such hybrid vector by an in vitro synthesis.
Claim 2
Dependent Claim
31 words
A method according to claim 1 for the preparation of a hybrid vector comprising the approximately 3.5 kbp EcoRI/SalI L. lactis insert of the plasmid pUCRS, or a functional fragment thereof.
Claim 3
Dependent Claim
25 words
A method according to claim 1 for the preparation of a hybrid vector in which said functional fragment retains MSP promoter, signal and/or structural functions.
Claim 4
Dependent Claim
22 words
A method according to claim 3 for the preparation of a hybrid vector in which said functional fragment retains MSP promoter function.
Claim 5
Dependent Claim
42 words
A method according to claim 4 for the preparation of a hybrid vector in which the functional fragment retaining promoter function extends from the start codon of the pre-MSP sequence up to a base in about position -1500 of the MSP gene.
Claim 6
Dependent Claim
45 words
A method according to claim 5 for the preparation of a hybrid vector in which the functional fragment retaining promoter function extends from the start codon of the pre-MSP sequence up to a base in about position -100 up to -1000 of the MSP gene.
Claim 7
Dependent Claim
26 words
A method according to claim 3 for the preparation of a hybrid vector in which said functional fragment retains the function of the MSP signal sequence.
Claim 8
Dependent Claim
32 words
A method according to claim 7 for the preparation of a hybrid vector in which the functional fragment retaining the MSP signal function encodes the 27 amino acid long MSP signal sequence.
Claim 9
Dependent Claim
45 words
A method according to claim 7 for the preparation of a hybrid vector in which the functional fragment retaining the MSP signal function extends from a base in about position 411 of the DNA sequence with SEQ ID No. 1 up to about position 491.
Claim 10
Dependent Claim
31 words
A method according to claim 3 for the preparation of a hybrid vector in which said functional fragment retains the MSP promoter function and the function of the MSP signal sequence.
Claim 11
Dependent Claim
106 words
A method according to claim 10 for the preparation of a hybrid vector in which the functional fragment retaining both the MSP promoter function and the function of the MSP signal sequence is selected from the group of fragments extending from any base between the EcoRI cut end of the approximately 3.5 kbp EcoRI/SalI L. lactis insert of the plasmid pUCRS and the first HindIII restriction site located in said insert upstream of the base corresponding to position 1 of the DNA sequence with SEQ ID No. 1. up to about the base corresponding to position 491 of the DNA sequence with SEQ ID No. 1.
Claim 12
Dependent Claim
26 words
A method according to claim 3 for the preparation of a hybrid vector in which said functional fragment retains the function of the MSP structural gene.
Claim 13
Dependent Claim
48 words
A method according to claim 12 for the preparation of a hybrid vector in which the functional fragment retaining the function of the MSP structural gene extends from a base in about position 492 up to about position 1793 of the DNA sequence with SEQ ID No. 1.
Claim 14
Dependent Claim
58 words
A method according to claim 1 for the preparation of a hybrid vector according to claim 1 comprising a DNA sequence which hybridizes with the approximately 3.5 kbp EcoRI/SalI L. lactis insert of the plasmid pUCRS or with a functional fragment thereof, or comprises a promoter region which is naturally operatively linked to such a hybridizing DNA sequence.
Claim 15
Dependent Claim
43 words
A method according to claim 14 for the preparation of a hybrid vector comprising a DNA sequence which encodes a gene related to the MSP gene or a functional fragment thereof or a promoter region of a gene related to the MSP gene.
Claim 16
Dependent Claim
52 words
A method according to claim 1 for the preparation of a hybrid vector comprising a DNA sequence which which encodes a signal peptide of MSP or a related peptide and which is degenerate in accordance with the meaning of the genetic code to a DNA sequence according to claim 1a) or 1b).
Claim 17
Dependent Claim
27 words
A method according to claim 1 for the preparation of a hybrid vector comprising a derivative of a DNA molecule according to claim 1a), 1b) or 1c).
Claim 18
Dependent Claim
63 words
A method according to claim 1 for the preparation of a hybrid vector comprising the origin of replication of (I) the 2.5 Md plasmid of L. lactis 712, or of (II) the 5.2 Md plasmid of L. lactis 712, or of (III) a plasmid of the same incompatibility group as the 2.5 Md plasmid or the 5.2 Md plasmid of L. lactis 712.
Claim 19
Dependent Claim
36 words
A method according to claim 18 for the preparation of a hybrid vector comprising the origin of replication located on the approximately 1.8 kbp long NdeI/SphI fragment of the 2.5 Md plasmid of L. lactis 712.
Claim 20
Dependent Claim
34 words
A method according to claim 18 for the preparation of a hybrid vector comprising the origin of replication located on the 1.0 kbp NdeI/HindIII fragment of the 5.2 Md plasmid of L. lactis 712.
Claim 21
Dependent Claim
44 words
A method according to claim 1 for the preparation of a hybrid vector which is selected from the group of vectors consisting of pUCRS, pSC12, pSC12ΔP, pSC12ΔN, pSC12ΔNP, pSC18, pSC18ΔP, or pSC18ΔN, pSC12HIR, pSC12HIR-1, pSC12HIRTerm, pUCRS, M13 mp 18RS, M13 mp19H, pVAHIR-1 and pVAHIR.
Claim 22
Dependent Claim
16 words
A method according to claim 1 for the preparation of a hybrid vector which is pSC12HIRTerm.
Claim 23
Dependent Claim
47 words
A method according to claim 1 for the preparation of a hybrid vector comprising a homologous or heterologous structural gene which is operatively linked in a proper reading frame to the DNA sequence encoding the MSP signal peptide and to the MSP promoter or a heterologous promoter.
Claim 24
Dependent Claim
179 words
A process for the prepararion of a DNA molecule which a) is the approximately 3.5 kbp EcoRI/SalI L. lactis insert of the plasmid pUCRS, or a functional fragment thereof, or b) hybridizes with said insert or with a functional fragment thereof, or comprises a promoter region which is naturally operatively linked to such a hybridizing DNA sequence, or c) is a degenerate sequence of a DNA sequence which is covered in a) or b) and which encodes a signal peptide, or d) is a derivative of a DNA molecule covered in a), b) or c), and/or e) comprises the origin of replication of (I) the 2.5 Md plasmid of L. lactis 712, or of (II) the 5.2 Md plasmid of L. lactis 712, or of (III) a plasmid of the same incompatibility group as the 2.5 Md plasmid or the 5.2 Md plasmid of L. lactis 712, said method comprising A) culturing a host which comprises such DNA molecule and isolating such DNA molecule from said cultured host, or B) preparing such DNA molecule by an in vitro synthesis.
Claim 25
Dependent Claim
52 words
A method for the preparation of a host transformed with a hybrid vector prepared according to claim 1, said method comprising the steps of (a) treating a host under transforming conditions with a hybrid vector prepared according to claim 1, optionally together with a selection marker gene, and (b) selecting the transformants.
Claim 26
Dependent Claim
77 words
A method according to claim 25 for the preparation of a transformed host selected from the group of transformed hosts consisting of E. coli strain TG1, C600 and HB101 transformed with either of the plasmids pUCRS, pSC12, pSC12ΔP, pSC12ΔN, pSC12ΔNP, pSC18, pSC18ΔN, pSC18ΔP, PSC12HIR, pSC12HIR-1, or pSC12HIRTerm, and L. cremoris , a plasmid-free L. lactis and a Bacillus strain transformed with either of pUCRS, pSC12, pSC12ΔP, pSC12ΔN, pSC12ΔNP, pSC18, pSC18ΔN, pSC18ΔP, pVAHIR, pVAHIR-1, pSC12HIR, pSC12HIR-1 or pSC12HIRTerm.
Claim 27
Dependent Claim
46 words
A method according to claim 25 for the preparation of a transformed host selected from the group of transformed hosts consisting of E. coli TG1 transformed with pUCRS, L. lactis transformed with pUCRS, pSC12 HIRTerm, pSC12 or pSC18, and B. thuringiensis transformed with pVAHIR or pVAHIR-1.
Claim 28
Independent Claim
77 words
A method for the preparation of a polypeptide comprising the steps of (a) fusing a homologous or heterologous structural gene in proper reading frame with the DNA sequence coding for the MSP signal peptide, (b) transforming a suitable host with a hybrid vector comprising a fused gene prepared in (a), (c) secreting the polypeptide encoded by said fused gene from the host cells and, when required, (d) isolating the polypeptide from the supernatant according to conventional methods.
Claim 29
Dependent Claim
26 words
A method according to claim 28 in which a host selected from the group of hosts consisting of L. lactis LM0230 and Bacillus spec. is used.
Claim 30
Dependent Claim
28 words
A method according to claim 28 in which a bacterial host is used which does not secrete in its culture medium a protease which degrades the expressed protein.
Claim 31
Dependent Claim
11 words
A method according to claim 28 in which desulfatohirudin is produced.
Claim 32
Dependent Claim
19 words
A method according to claim 31 in L. lactis LM0230 cells transformed with pSC12HIR, pSC12HIR-1 or pSC12HIRTerm are used.
Claim 33
Dependent Claim
15 words
A method according to claim 31 in L. lactis LM0230 cells transformed pSC12HIRTerm are used.
Claim 34
Dependent Claim
18 words
A method according to claim 31 in which B. thurinpiensis cells transformed with pVAHIR or pVAHIR-1 are used.
Claim 35
Dependent Claim
62 words
A method according to claim 28 characterized in that (a) the transformed cells are collected from the nutrient medium either in the log phase or in the stationary phase of the culture, (b) they are resuspended in a smaller volume of fresh nutrient medium or suitable buffer solution and (c) they are further incubated before isolating the desired product from the supernatant.
Claim 36
Dependent Claim
27 words
A method according to claim 35 characterized in that the transformed cells are resuspended in about 1 % up to 20 % of the original culture volume.
Claim 37
Dependent Claim
36 words
A method according to claim 36 characterized in that the L. lactis LM0230 cells transformed with pSC12HIRTerm are resuspended in about 1/10 vol. of fresh nutrient medium and incubated for about 30 min at about 30°C.
Claim 38
Dependent Claim
11 words
A method according to claim 28 in which MSP is produced.
Claim 39
Independent Claim
54 words
A method for the production of pure MSP comprising the steps of (a) precipitating the proteins from the supernatant of L. lactis LM0230 with trichloroacetic acid, (b) electrophoresis of the precipitated protein on a SDS polyacrylamide gel, (c) cutting out the region with the major protein band and (d) electroeluting MSP from the gel.
References Cited (Backward Citations)
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