Claims
- 1. A process for the production of a peptide, polypeptide, or protein having a predetermined property, comprising the steps of:
producing by synthetic polynucleotide coupling, stochastically generated polynucleotide sequences; forming a library of expression vectors containing such stochastically generated polynucleotide sequences; culturing host cells containing the vectors to produce peptides, polypeptides, or proteins encoded by the stochastically generated polynucleotide sequences; carrying out screening or selection on such host cells, to identify a peptide, polypeptide, or protein produced by the host cells having the predetermined property; isolating a stochastically generated polynucleotide sequence which encodes the identified peptide, polypeptide, or protein; using the isolated sequence to produce the peptide, polypeptide, or protein having the predetermined property.
- 2. A process for the production of a peptide, polypeptide, or protein having a predetermined property, comprising the steps of:
producing at least partially stochastic synthetic polynucleotide sequences, introducing the at least partially stochastic polynucleotide sequences thus obtained into host cells, cultivating the transformed host cells containing these at least partially stochastic polynucleotide sequences so as to clone the stochastic polynucleotide sequences and lead to the production of peptides, polypeptides, or proteins expressed by at least some of these stochastic polynucleotide sequences, carrying out screening and/or selection methods on such clones of transformed host cells to identify those clones producing the peptide, polypeptide, or protein having the predetermined property, isolating the clones so identified, and growing the isolated clones in a manner so as to produce the peptide, polypeptide, or protein having the predetermined property.
- 3. The process according to claim 2 wherein the at least partially stochastic polynucleotide sequences are introduced into host cells by an expression vector.
- 4. A process for the production of a peptide, polypeptide, or protein having a predetermined property by microbiological means, comprising the steps of:
simultaneously producing in a common milieu expression vectors which include at least one stochastic sequence of polynucleotides, introducing the expression vectors thus obtained into host cells, cultivating the host cells containing these expression vectors so as to clone the stochastic sequences and lead to the production of peptides, polypeptides, or proteins expressed by at least some of these stochastic sequences, carrying out screening and/or selection methods on such clones to identify those clones producing the peptide, polypeptide, or protein having the predetermined property, isolating the clones so identified, and growing the isolated clones in a manner so as to produce the peptide, polypeptide, or protein having the predetermined property.
- 5. A process for the production of a peptide, polypeptide, or protein having a predetermined property by microbiological means, comprising the steps of:
simultaneously producing in a common milieu expression vectors which include at least one stochastic sequence of polynucleotides, introducing the expression vectors thus obtained into host cells, cultivating the host cells containing these expression vectors so as to clone the expression vector and lead to the production of peptides, polypeptides, or proteins expressed by at least some of these expression vectors, carrying out screening and/or selection methods on such clones producing the peptide, polypeptide, or protein having the predetermined property, isolating the clones so identified, and growing the isolated clones in a manner so as to produce the peptide, polypeptide, or protein having the predetermined property.
- 6. A process for the production of a peptide, polypeptide, or protein having a predetermined property, comprising the steps of:
producing a library of at least partially stochastic synthetic polynucleotide sequences, amplifying and translating these at least partially stochastic polynucleotide sequences to produce peptides, polypeptides, or proteins expressed by at least some of these stochastic polynucleotide sequences, and carrying out screening and/or selection methods to obtain the peptide, polypeptide, or protein having at least the predetermined property.
- 7. The process according to claim 6 wherein the at least partially stochastic polynucleotide sequences undergo extracellular amplification.
- 8. The process according to claim 1 wherein polynucleotide sequences are produced by stochastic copolymerization of desired ratios of the deoxyphosphonucleotides guanine, cytosine, thymidine, and adenine, starting from the two extremities of an expression vector which was previously linearized, then by formation of cohesive extremities to create a first strand of stochastic DNA constituting a molecule of expression vector possessing two stochastic sequences whose 3′ extremities are complementary, followed by synthesis of the second strand of the stochastic DNA.
- 9. The process according to claim 1 wherein the polynucleotide sequences are produced by stochastic copolymerization of double stranded oligonucleotides which do not have cohesive ends, in a manner so as to form fragments of stochastic DNA, followed by ligation of these fragments in an expression vector which was previously linearized.
- 10. The process according to claim 1 wherein the host cells are prokaryotic cells.
- 11. The process according to claim 10 wherein the prokaryotic cells are HB 101 or C 600.
- 12. The process according to claim 1 wherein the host cells are eukaryotic cells.
- 13. The process according to claim 1 wherein the expression vector is a plasmid.
- 14. The process according to claim 13 wherein the plasmid is pUC8.
- 15. The process according to claim 1 wherein the expression vector is viral DNA.
- 16. The process according to claim 1 wherein the expression vector is a hybrid of plasmid and viral DNA.
- 17. The process according to claim 1 wherein the expression vector is a phage.
- 18. The process according to claim 1 wherein during selection, the DNA which encodes for the peptide, polypeptide, or protein is mutagenized.
- 19. The process according to claim 18 wherein the DNA undergoes in vivo mutagenesis by recombination.
- 20. The process according to claim 18 wherein the DNA undergoes in vitro mutagenesis by mutagens.
- 21. The process according to claim 18 wherein the predetermined property is an adaptive improvement in the capacity to fulfill a desired function.
- 22. The process according to claim 1 wherein the predetermined property is the capacity to catalyse a given chemical reaction.
- 23. The process according to claim 1 wherein the predetermined property is the capacity to simulate at least one biologically active compound.
- 24. The process according to claim 23 wherein the biologically active compound is a member of the group consisting of hormones, neurotransmitters, growth factors, and specific regulators of replication, transcription, or translation of nucleic acids.
- 25. The process according to claim 1 wherein the predetermined property is the capacity to modify at least one biological function of at least one biologically active compound.
- 26. The process according to claim 25 wherein the biologically active compound is a member of the group consisting of hormones, neurotransmitters, growth factors, and specific regulators of replication, transcription, or translation of nucleic acids.
- 27. The process according to claim 1 wherein the predetermined property is that of having at least one epitope similar to one of the epitopes of a given antigen.
- 28. The process according to claim 27 wherein the antigen is epidermal growth factor.
- 29. The process according to claim 27 wherein the said property is the capacity to simulate an epitope of a given antigen, and that screening and/or selection of clones producing a peptide, polypeptide, or protein having this property is carried out by obtaining molecules which bind to that epitope, utilizing these molecules so obtained to identify those clones containing the peptide, polypeptide, or protein, growing the clones thus identified, separating and purifying the peptide, polypeptide, or protein produced by the clones, and then submitting these peptide(s), polypeptide(s), or protein(s) to an in vitro assay to verify that it has the said property.
- 30. The process according to claim 27 wherein the peptide, polypeptide, or protein having said property is used to make a vaccine.
- 31. The process according to claim 27 wherein an ensemble of peptides, polypeptides, or proteins having said property is used to make a vaccine.
- 32. The process according to claim 1 wherein the clones producing a peptide, polypeptide, or protein having the predetermined property are identified and isolated by affinity chromatography on antibodies corresponding to a protein expressed by a natural fragment of DNA.
- 33. The process according to claim 32 wherein after expression and purification of the identified peptide, polypeptide, or protein, novel fragments of the peptide, polypeptide, or protein having the predetermined property are separated and isolated.
- 34. The process according to claim 32 wherein the natural fragment of DNA contains a gene expressing B-galactosidase, and that the peptide, polypeptide, or protein is identified and isolated by affinity chromatography with anti-β-galactosidase antibodies.
- 35. The process according to claim 34 wherein after expression and purification of the identified peptide, polypeptide, or protein, novel fragments of the peptide, polypeptide, or protein having the predetermined property are separated and isolated.
- 36. The process according to claim 1 wherein the predetermined property is the capacity to bind to a given compound.
- 37. The process according to claim 36 wherein the said given compound is a member of the group consisting of peptides, polypeptides, and proteins.
- 38. The process according to claim 36 wherein the said given compound is a member of the group consisting of sequences of DNA and RNA.
- 39. The process according to claim 38 wherein the sequence of DNA acts as a cis-regulatory sequence of replication or transcription of a neighboring sequence of DNA.
- 40. The process according to claim 1 wherein the number of different peptides, polypeptides, or proteins expressed by all of the at least partially stochastic polynucleotide sequences is greater than 10,000.
- 41. The process according to claim 2 wherein the number of different peptides, polypeptides, or proteins expressed by all of the at least partially stochastic polynucleotide sequences is greater than 10,000.
- 42. The process according to claim 6 wherein the number of different peptides, polypeptides, or proteins expressed by all of the at least partially stochastic polynucleotide sequences is greater than 10,000.
- 43. The process according to claim 1 wherein DNA having the predetermined property is isolated from the clones.
- 44. The process according to claim 1 wherein RNA having the predetermined property is isolated from the clones.
- 45. A process for the production of an expression vector which includes at least one stochastic sequence of polynucleotides, comprising the steps of:
providing, in a common milieu, at least three oligonucleotides, said oligonucleotides comprising at least 7 nucleotides, polymerizing said nucleotides to form stochastic double stranded DNA fragments, and ligating said stochastic double stranded DNA fragments into an expression vector.
- 46. The process according to claim 45 wherein the oligonucleotides are heptamers.
- 47. The process according to claim 46 wherein the transforming DNA derived from a culture of clones is isolated and purified, the purified DNA is cut by means of at least one restriction enzyme which corresponds to a specific restriction site present in the palindromic heptamers or octamers but absent from the expression vector, then the ensemble of linearized stochastic DNA fragments obtained are simultaneously treated with T4 ligase in a manner to create a new ensemble of DNA containing new stochastic sequences, and this new ensemble of DNA is used to transform host cells.
- 48. The process according to claim 46 wherein the heptamers are palindromic.
- 49. The process according to claim 48 wherein the palindromic heptamers are selected from the group consisting of:
- 50. The process according to claim 46 wherein the oligonucleotides are octamers.
- 51. The process according to claim 50 wherein the transforming DNA derived from a culture of clones is isolated and purified, the purified DNA is cut by means of at least one restriction enzyme which corresponds to a specific restriction site present in the palindromic heptamers or octamers but absent from the expression vector, then the ensemble of linearized stochastic DNA fragments obtained are simultaneously treated with T4 ligase in a manner to create a new ensemble of DNA containing new stochastic sequences, and this new ensemble of DNA is used to transform host cells.
- 52. The process according to claim 50 wherein the octamers are palindromic.
- 53. The process according to claim 52 wherein the palindromic octamers are selected from the group consisting of:
- 54. The process according to claim 45 wherein the stochastic double stranded DNA fragments are about 160 to 800 base pairs in length.
- 55. A process for the production of an expression vector which includes at least one stochastic sequence of polynucleotides, comprising the steps of:
linearizing an expression vector, reacting said linearized vector with terminal transferase in the presence of desired ratios of deoxynucleotide-triphosphates of guanine, cytosine, thymidine, and adenine to form polymers, hybridizing said polymers, and treating said polymers to form expression vectors consisting of stochastic double stranded DNA.
- 56. A process for the production of an expression vector comprising the steps of:
producing a library expression vectors which include at least one stochastic synthetic sequence of polynucleotides, introducing the expression vectors thus obtained into host cells, cultivating the host cells containing these expression vectors so as to clone the expression vector and lead to the production of peptides, polypeptides, or proteins expressed by at least some of these expression vectors, carrying out screening and/or selection methods on such clones producing the peptide, polypeptide, or protein having the predetermined property, and isolating the cloned expression vectors so identified.
- 57. A process for the production of a peptide, polypeptide, or protein having a predetermined property, comprising the steps of:
producing by synthetic polynucleotide coupling, stochastically generated polynucleotide sequences; forming a library of expression vectors containing such stochastically generated polynucleotide sequences; culturing host cells containing the vectors to produce peptides, polypeptides, or proteins encoded by the stochastically generated polynucleotide sequences; carrying out screening or selection on such host cells, to identify a peptide, polypeptide, or protein produced by the host cells having the predetermined property.
- 58. A process for the production of a peptide, polypeptide, or protein having a predetermined property, comprising the steps of:
producing at least partially stochastic polynucleotide sequences, introducing the at least partially stochastic polynucleotide sequences thus obtained into host cells, cultivating the transformed host cells containing these at least partially stochastic polynucleotide sequences so as to clone the stochastic polynucleotide sequences and lead to the production of peptides, polypeptides, or proteins expressed by at least some of these stochastic polynucleotide sequences, carrying out screening and/or selection methods on such clones of transformed host cells to identify the peptide, polypeptide, or protein having the predetermined property.
- 59. A process for the production of a host cell containing an expression vector which includes at least one stochastic sequence of polynucleotides, comprising the steps of:
producing a library of expression vectors which include at least one stochastic sequence of polynucleotides, said expression vectors being produced by the following steps:
providing, in a common milieu, at least three oligonucleotides, said oligonucleotides comprising at least 7 nucleotides; polymerizing said nucleotides to form stochastic double stranded DNA fragments; and ligating said stochastic double stranded DNA fragments into an expression vector; introducing the expression vectors thus obtained into host cells; and cultivating the host cells containing these expression vectors so as to clone the stochastic sequences and lead to the production of peptides, polypeptides, or proteins expressed by at least some of these stochastic sequences.
- 60. A process for the production of a host cell containing an expression vector which includes at least one stochastic sequence of polynucleotides, comprising the steps of:
producing a library of expression vectors which include at least one stochastic sequence of polynucleotides, said expression vectors being produced by the following steps:
linearizing an expression vector, reacting said linearized vector with terminal transferase in the presence of desired ratios of deoxynucleotide-triphosphates of guanine, cytosine, thymidine, and adenine to form polymers, hybridizing said polymers, and treating said polymers to form expression vectors consisting of stochastic double stranded DNA; introducing the expression vectors thus obtained into host cells; and cultivating the host cells containing these expression vectors so as to clone the stochastic sequences and lead to the production of peptides, polypeptides, or proteins expressed by at least some of these stochastic sequences.
- 61. A process for the production of a host cell containing an expression vector which includes at least one stochastic sequence of polynucleotides, comprising the steps of:
producing a library of expression vectors which include at least one stochastic synthetic sequence of polynucleotides, introducing the expression vectors thus obtained into host cells, cultivating the host cells containing these expression vectors so as to clone the stochastic sequences and lead to the production of peptides, polypeptides, or proteins expressed by at least some of these stochastic sequences, carrying out screening and/or selection methods on such clones to identify those clones producing the peptide, polypeptide, or protein having the predetermined property, and isolating the cloned host cells so identified.
- 62. A library of expression vectors comprising stochastic polynucleotide sequences which encode at least 10,000 peptides, polypeptides, or proteins.
- 63. An expression vector produced in accordance with the process of claim 45, 55, or 56.
- 64. A host cell produced in accordance with the process of claim 59, 60, or 61.
- 65. The peptide, polypeptide, or protein produced in accordance with claim 1, 2, 4, 5, 6, 57, or 58.
- 66. A process for the production of an ensemble of peptides, polypeptides, or proteins, which ensemble has the capacity to catalyze a sequence of chemical reactions to thereby produce a desired compound, comprising the steps of:
a. producing a library of at least partially stochastic polynucleotide sequences, b. introducing the at least partially stochastic polynucleotide sequences thus obtained into host cells, c. simultaneously cultivating the independent transformed host cells containing these at least partially stochastic polynucleotide sequences so as to clone the stochastic polynucleotide sequences and lead to the production of a first set of peptides, polypeptides, or proteins expressed by at least some of these stochastic polynucleotide sequences, d. combining the first set of peptides, polypeptides, or proteins with a collection of precursors to said desired compound under conditions favorable for said sequence of chemical reactions; e. determining if said desired compound is produced, and if so, dividing the first set of peptides, polypeptides, or proteins into a plurality of subsets; f. combining each of the subsets with the collection of precursors under conditions favorable for said sequence of chemical reactions; g. determining, subset by subset, if said desired compound is produced with each of said subsets, and if so, dividing that subset into further subsets; and h. repeating steps f. and g. until the ensemble of peptides, polypeptides, or proteins which catalyze the sequence of chemical reactions to produce the desired compound is identified.
Priority Claims (1)
Number |
Date |
Country |
Kind |
1379/85-8 |
Mar 1985 |
CH |
|
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation of U.S. patent application Ser. No. 942,630 filed Nov. 20, 1986, the entire disclosure of which is incorporated herein by reference.
Continuations (7)
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08464569 |
Jun 1995 |
US |
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10138213 |
May 2002 |
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08349510 |
Dec 1994 |
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08464569 |
Jun 1995 |
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08133952 |
Oct 1993 |
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08349510 |
Dec 1994 |
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07977307 |
Nov 1992 |
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08133952 |
Oct 1993 |
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07616319 |
Nov 1990 |
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07977307 |
Nov 1992 |
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06942630 |
Nov 1986 |
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07616319 |
Nov 1990 |
US |
Parent |
PCT/CH85/00099 |
Jun 1985 |
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06942630 |
Nov 1986 |
US |