Patent US5783431A successfully refreshed from Google Patents.
US5783431A
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Methods for generating and screening novel metabolic pathways
Assignee: Merck Sharp & Dohme LLC
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Filing: 1996-10-24
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Granted: 1998-07-21
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Expires: 2016-10-24
Abstract
Abstract The present invention relates to a novel drug discovery system for generating and screening molecular diversity. The system provides methods for mixing and cloning genetic materials from a plurality of species of organisms in combinatorial gene expression libraries to generate novel metabolic pathways and classes of compounds. The system also provides mobilizable combinatorial gene expression libraries that can be transferred from one species of host organism to another for expression. Also provided are specialized cloning vectors for making mobilizable gene expression libraries. The system also involves methods for pre-screening or identifying for host organisms containing a library that are capable of generating such novel pathways and compounds.
Inventors
- 👤 Todd C. Peterson
- 👤 Lyndon M. Foster
- 👤 Paul Brian
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 18:36
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6811; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/64; C12N15/10; C12N15/102; C12N15/1027; C12N15/1034; C12N15/66; C12N15/79; C12N15/80; C12N15/81
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
99 words
A mobilizable combinatorial gene expression library, comprising a pool of expression constructs, each expression construct comprising a shuttle vector that replicates in different species or strains of host cell, said shuttle vector containing one or more cDNA or genomic DNA fragments, wherein the cDNA or genomic DNA fragments in the pool of expression constructs are derived from a plurality of species of donor organisms, and wherein the cDNA or genomic DNA fragments are each operably-associated with one or more regulatory regions that drives expression of genes encoded by the cDNA or genomic DNA fragments in an appropriate host organism.
Claim 2
Dependent Claim
99 words
A mobilizable combinatorial gene expression library, comprising a pool of expression constructs, each expression construct comprising a shuttle vector that replicates in different species or strains of host cell, said shuttle vector containing one or more cDNA or genomic DNA fragments, wherein the cDNA or genomic DNA fragments in the pool of expression constructs are derived from a plurality of species of donor organisms, and wherein the cDNA or genomic DNA fragments are each operably-associated with one or more regulatory regions that drives expression of genes encoded by the cDNA or genomic DNA fragments in an appropriate host organism.
Claim 3
Dependent Claim
33 words
The gene expression library of claim 1 wherein the cDNA or genomic DNA fragments contained in the expression constructs are randomly concatenated, and are derived from one or more species of donor organisms.
Claim 4
Dependent Claim
25 words
The gene expression library of claim 1 wherein the cDNA or genomic DNA fragments contained in the expression constructs are preselected for a specific property.
Claim 5
Dependent Claim
32 words
The gene expression library of claim 1, 2, or 3 in which the expression construct comprises a plasmid vector, a phage vector, a viral vector, a cosmid vector, or an artificial chromosome.
Claim 6
Dependent Claim
24 words
The gene expression library of claim 4 in which the vector is a shuttle vector that replicates in different host cell species or strains.
Claim 7
Dependent Claim
20 words
The gene expression library of claim 1, 2, or 3 in which the donor organisms comprise a mixture of microorganisms.
Claim 8
Dependent Claim
21 words
The gene expression library of claim 1, 2, or 3 in which each expression construct is contained in a host cell.
Claim 9
Dependent Claim
37 words
The gene expression library of claim 7 in which the host cells have been modified by the introduction, induction or overproduction of a known metabolic pathway of interest or portion thereof prior to containing the expression constructs.
Claim 10
Dependent Claim
48 words
The gene expression library of claim 7 in which the host cell is Escherichia coli, Bacillus subtilis, Streptomyces lividans, Streptomyces coelicolor, Pseudomonas aeruginosa, Myxococcus xanthus, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Spodoptera frugiperda, Aspergillus nidulans, Arabidopsis thaliana, Nicotiana tabacum, COS cells, 293 cells, VERO cells, NIH/3T3 cells, or CHO cells.
Claim 11
Dependent Claim
34 words
The gene expression library of claim 7 in which the host cells further contain a reporter regimen tailored to identify clones in the library that are expressing desirable metabolic pathways, gene products, or compounds.
Claim 12
Dependent Claim
42 words
The gene expression library of claim 7 in which the reporter regimen comprises DNA encoding a reporter gene operably-associated with a regulatory region that is inducible or modulated by the desirable metabolic pathways, gene products, or compounds expressed by the host cell.
Claim 13
Dependent Claim
37 words
The gene expression library of claim 7 in which the host cells are in a matrix containing a reporter regimen tailored to identify clones in the library that are expressing desirable metabolic pathways, gene products, or compounds.
Claim 14
Independent Claim
97 words
A method for making a mobilizable combinatorial gene expression library, comprising ligating a shuttle vector that replicates in different species or strains of host cell, to one or more cDNA or genomic DNA fragments to form a pool of expression constructs, wherein said cDNA or genomic DNA fragments in the pool of expression constructs are obtained from a plurality of species of donor organisms, and wherein the genes contained in the cDNA or genomic DNA fragments are each operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.
Claim 15
Independent Claim
97 words
A method for making a mobilizable combinatorial gene expression library, comprising ligating a shuttle vector that replicates in different species or strains of host cell, to one or more cDNA or genomic DNA fragments to form a pool of expression constructs, wherein said cDNA or genomic DNA fragments in the pool of expression constructs are obtained from a plurality of species of donor organisms, and wherein the genes contained in the cDNA or genomic DNA fragments are each operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.
Claim 16
Dependent Claim
31 words
The method of claim 13 wherein the cDNA or genomic DNA fragments contained in the expression constructs are randomly concatenated, and are derived from one or more species of donor organisms.
Claim 17
Dependent Claim
23 words
The method of claim 13 wherein the cDNA or genomic DNA fragments contained in the expression constructs are preselected for a specific property.
Claim 18
Dependent Claim
30 words
The method of claim 13, 14, or 15 in which the DNA vector is a plasmid vector, a phage vector, a viral vector, a cosmid vector, or an artificial chromosome.
Claim 19
Dependent Claim
19 words
The method of claim 13, 14, or 15 in which the shuttle vector further comprises an origin of transfer.
Claim 20
Independent Claim
115 words
A method for making a combinatorial gene expression library comprising transferring a pool of expression constructs in a species of host organism to another species or strain of host organism, said expression construct comprising a shuttle vector that replicates in different species or strains of host cell, said shuttle vector comprising one or more cDNA or genomic DNA fragments, wherein the cDNA or genomic DNA fragments in the pool of expression constructs are obtained from a plurality of species of donor organisms, and wherein the genes contained in the cDNA or genomic DNA fragments are each operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.
Claim 21
Independent Claim
115 words
A method for making a combinatorial gene expression library comprising transferring a pool of expression constructs in a species of host organism to another species or strain of host organism, said expression construct comprising a shuttle vector that replicates in different species or strains of host cell, said shuttle vector comprising one or more cDNA or genomic DNA fragments, wherein the cDNA or genomic DNA fragments in the pool of expression constructs are obtained from a plurality of species of donor organisms, and wherein the genes contained in the cDNA or genomic DNA fragments are each operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.
Claim 22
Dependent Claim
15 words
The method of claim 18 wherein the pool of expression constructs is transferred by conjugation.
Claim 23
Dependent Claim
39 words
The method of claim 18 wherein the pool of expression constructs is transferred by isolating the expression constructs from a first species of host organism, and introducing the expression constructs into a second species or strain of host organism.
Claim 24
Independent Claim
90 words
A method for making a biased combinatorial gene expression library, comprising ligating a DNA vector to one or more cDNA or genomic DNA fragments to generate a library of expression constructs, wherein the cDNA or genomic DNA fragments in the library are obtained from a plurality of species of donor organisms and are selected for a specific property, and wherein genes contained in the cDNA or genomic DNA fragments are each operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.
Claim 25
Independent Claim
90 words
A method for making a biased combinatorial gene expression library, comprising ligating a DNA vector to one or more cDNA or genomic DNA fragments to generate a library of expression constructs, wherein the cDNA or genomic DNA fragments in the library are obtained from a plurality of species of donor organisms and are selected for a specific property, and wherein genes contained in the cDNA or genomic DNA fragments are each operably-associated with their native or exogenous regulatory regions which drive expression of the genes in an appropriate host cell.
Claim 26
Dependent Claim
28 words
The gene expression library of claim 3 wherein the cDNA or genomic DNA fragments are preselected for homology to nucleic acid sequences encoding proteins in a metabolic pathway.
Claim 27
Dependent Claim
26 words
The method of claim 15 wherein the cDNA or genomic DNA fragments are preselected for homology to nucleic acid sequences encoding proteins in a metabolic pathway.
Claim 28
Dependent Claim
33 words
The gene expression library of claim 1, 2, or 3 in which each expression construct is contained in a host cell that contains proteins that mediate transfer of the expression construct by conjugation.
Claim 29
Dependent Claim
28 words
The method of claim 18 wherein the pool of expression constructs is contained in host cells that contain proteins that mediate transfer of the expression constructs by conjugation.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2001-07-20 | 2002-01-19 | 2002-07-21 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-07-20 | 2006-01-19 | 2006-07-21 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-07-20 | 2010-01-19 | 2010-07-21 | $7700.00 | Window Closed / Lapsed |