Process for the production of stochastically-generated transcription or translation products

• US 5,814,476 A
• Filed: 06/05/1995
• Issued: 09/29/1998
• Est. Priority Date: 03/30/1985
• Status: Expired due to Term

• Current Assignee: Stuart Kauffman
• Original Assignee: Stuart Kauffman
• Inventors: Ballivet, Marc, Kauffman, Stuart Alan
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): Wai, Thanda
• Application Number:
  US08/468,477
• Time in Patent Office: 1,212 Days
• Field of Search: 435/7.1, 435/69.1, 435/91.1, 435/172.3, 435/320.1, 536/23.1, 530/300, 530/350
• US Class Current: 435/69.1
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/485 Epidermal growth factor [EGF], i.e. urogastrone
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 15/68 Stabilisation of the vector
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 2730/10122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 9/2471 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase

Abstract

The present invention is directed to a process for the production of a peptide, polypeptide, or protein having a predetermined property. In accordance with one embodiment, the process begins by producing by way of synthetic polynucleotide coupling, stochastically generated polynucleotide sequences. A library of expression vectors containing such stochastically generated polynucleotide sequences is formed. Next, host cells containing the vectors are cultured so as to produce peptides, polypeptides, or proteins encoded by the stochastically generated polynucleotide sequences. Screening or selection is carried out on such host cells to identify a peptide, polypeptide, or protein produced by the host cells which has the predetermined property. The stochastically generated polynucleotide sequence which encodes the identified peptide, polypeptide, or protein is then isolated and used to produce the peptide, polypeptide, or protein having the predetermined property.

Claims

1. A process for the production of a transcription product or a translation product, comprising the steps of:

producing a stochastically-generated polynucleotide sequence;

producing a library of expression vectors comprising said stochastic polynucleotide sequence;

transforming or transfecting a competent clone with said library of expression vectors;

amplifying said transformed or transfected competent clone;

screening and/or selecting said transformed or transfected clone in order to isolate a clone expressing a stochastic polynucleotide sequence capable of synthesizing a transcription product or a translation product having a predetermined property; and

isolating said selected or screened transformed clone;

isolating a stochastically generated polynucleotide sequence which encodes the identified transcription product or translation product

using the isolated sequence to produce the transcription product or translation product having the predetermined property.

12. The process according to claim 1 or 2 wherein said translation product comprises a product having a desired property and is selected from the group consisting of a peptide, a polypeptide or a protein.

13. The process according to claim 1 or 2 wherein said transcription product comprises a product having a desired property and is selected from the group consisting of a RNA or a DNA.

14. A library of vectors produced by the process of claim 1 wherein said library comprises stochastic nucleotide sequences encoding for at least 10,000 transcription products or translation products.

2. A process for the production of a transcription product or a translation product, comprising the steps of:

producing a diverse population of stochastic polynucleotide sequences;

inserting said stochastic polynucleotide sequences into expression vectors to form a diverse population of expression vectors;

transforming or transfecting competent clones with said diverse population of expression vectors comprising said stochastic polynucleotide sequences;

amplifying said transformed or transfected competent clone;

screening and/or selecting said transformed or transfected clones in order to isolate a clone expressing a stochastic polynucleotide capable of synthesizing a transcription product or a translation product having the predetermined property;

isolating said selected or screened transformed clone;

isolating said stochastic polynucleotide sequence which encodes the identified transcription product or translation product;

using the isolated stochastic polynucleotide sequence so as to produce the transcription product or translation product having the predetermined property.

3. A process for the production of a polynucleotide comprising,

producing in an appropriate buffer a diverse population of stochastic polynucleotide sequences;

inserting said stochastic polynucleotide sequences into vectors to form a diverse population of vectors;

introducing said diverse population of vectors into host cells in a manner to produce a diverse population of transformed host cells;

producing independent clones of the host cells so produced;

screening and/or selecting said independent clones of the host cells to identify host cells comprising a stochastic polynucleotide sequence having at least one desired property; and

isolating said stochastic polynucleotide sequence from the selected or screened clones of host cells.

4. The process according to claim 3, wherein said stochastic polynucleotide sequence comprises a capacity to specifically bind to a compound.

5. The process according to claim 4, wherein said compound is selected from the group consisting of peptides, polypeptides and proteins.

6. The process according to claim 4, wherein said compound further comprises a compound regulating transcriptional activity or a compound regulating replication activity of DNA.

7. A process according to claim 6, wherein said compound further comprises a regulatory protein controlling the transcription or replication of DNA.

8. A process for the production of an RNA comprising,

producing in an appropriate buffer a diverse population of stochastic polynucleotide sequences;

inserting said stochastic polynucleotide sequences into vectors to form a diverse population of vectors;

introducing said diverse population of vectors into host cells in a manner to produce a diverse population of transformed host cells;

producing independent clones of transformed or transfected host cells;

screening and/or selecting said independent clones of the host cells to identify host cells comprising a stochastic polynucleotide sequence capable of producing RNA having at least one desired property; and

isolating said stochastic polynucleotide sequence from the selected or screened clones of host cells.

9. The process according to claim 8, wherein said RNA further comprises a capacity to specifically bind to a compound.

10. The process according to claim 8, wherein said RNA further comprises the capacity to catalyze a chemical reaction.

11. The process according to claim 8, wherein said RNA further comprises a transfer RNA.

15. A method of identifying a polynucleotide having a predetermined property, comprising:

(a) producing a population of polynucleotides comprising greater than about 1×

10⁵ different stochastic polynucleotide sequences;

(b) screening said population of polynucleotides for said predetermined property under conditions which allow detection of one or more polynucleotides having said predetermined property.

16. The method of claim 15, wherein step (a) further comprises synthesizing a population of at least partially stochastic polynucleotide sequences.

17. The method of claim 15 or 16, further comprising amplification of said population of stochastic polynucleotide sequences.

18. The method of claim 15 or 16, wherein step (a) further comprises expressing said population of stochastic polynucleotide sequences to produce said population of polynucleotides.

19. The method of claim 15 or 16, wherein said population of polynucleotides comprise DNA.

20. The method of claim 15 or 16, wherein said population polynucleotides comprise RNA.

21. The method of claim 15, further comprising isolating said one or more polynucleotide sequences having said predetermined property.

22. The method of claim 15, wherein said predetermined property comprises binding or chemical catalysis.

23. The method of claim 22, further comprising improving said predetermined property by in vitro or in vivo mutagenesis.

24. The method of claim 22, wherein said binding further comprises modification of a biological or chemical property of a compound bound by said polynucleotide.

25. The method of claim 24, wherein said modification of said biological or chemical property of said compound further comprises stimulating or inhibiting at least one biological function of said compound.

26. The method of claim 15, wherein said population of polynucleotides comprises greater than about 1×

10⁵ different stochastic polynucleotide sequences.

27. The method of claim 15, wherein said population of polynucleotides comprises greater than about 1×

10⁷ different stochastic polynucleotide sequences.

28. The method of claim 15, wherein said population of polynucleotides comprises greater than about 1×

10⁸ different stochastic polynucleotide sequences.

29. A method of identifying a polynucleotide having a binding property to a ligand, comprising:

(a) synthesizing a population of stochastic polynucleotide sequences;

(b) inserting said population of stochastic polynucleotide sequences into a population of vectors to form a population of vectors containing stochastic polynucleotide sequences;

(c) expressing in host cells said population of vectors containing stochastic polynucleotide sequences to produce a diverse population of expressed polynucleotides, and

(d) screening said diverse population of polynucleotides with a ligand under conditions which allow binding and detection one or more polynucleotides having said binding property to said ligand.

30. The method of claim 29, wherein said inserting further comprises hybridization of complementary ends.

31. The method of claim 29, wherein said inserting further comprises ligation.

32. The method of claim 29, therein said population of stochastic polynucleotide sequences are produced by stochastic copolymerization of oligonucleotides or polynucleotides.

33. The method of claim 29, wherein said population of stochastic polynucleotide sequences are produced by copolymerization of nucleotide triphosphates consisting of Adenine, Cytosine, Guanine, Thymine and Uracil.

34. The method of claim 29, wherein said population of stochastic polynucleotide sequences are produced by chemical synthesis.

35. The method of claim 29, wherein said population of stochastic polynucleotide sequences comprise DNA.

36. The method of claim 29, wherein said population of stochastic polynucleotide sequences comprise RNA.

37. The method of claim 29, wherein the vector is a plasmid.

38. The method of claim 29, wherein the vector is viral DNA.

39. The method of claim 29, wherein the vector is a hybrid of plasmid and viral DNA.

40. The method of claim 29, wherein the vector is a phage.

41. The method of claim 29, wherein step (c) further comprises digesting the population of vectors with a restriction enzyme having a recognition sequence absent in the vector and reinserting the digested products into said digested population of vectors to form a different population having a greater number of new stochastic polynucleotide sequences.

42. The method of claim 29, wherein said population of stochastic polynucleotide sequences, comprise at least 1×

10⁴ different polynucleotide sequences.

43. The method of claim 29, wherein said population of stochastic polynucleotide sequences, comprise greater than 1×

10⁵ different polynucleotide sequences.

44. The method of claim 29, wherein said population of stochastic polynucleotide sequences, comprise greater than 1×

10⁶ different polynucleotide sequences.

45. The method of claim 29, wherein said population of stochastic polynucleotide sequences, comprise greater than 1×

10⁷ different polynucleotide sequences.

46. The method of claim 29, wherein said population of stochastic polynucleotide sequences, comprise greater than 1×

10⁸ different polynucleotide sequences.

47. A method of isolating a polynucleotide having a predetermined property, comprising:

(a) producing a population of polynucleotides comprising greater than 1×

10⁵ different stochastic polynucleotide sequences;

(b) screening said population of stochastic polynucleotide sequences for said predetermined property under conditions which allow detection of one or more polynucleotides having said predetermined property, and

(c) isolating the one or more polynucleotide sequences having said predetermined property.

48. The method of claim 47, wherein step (a) further comprises synthesizing a population of at least partially stochastic polynucleotide sequences.

49. The method of claim 47 or 48, further comprising amplification of said population of stochastic polynucleotide sequences.

50. The method of claim 47 or 48, wherein step (a) further comprises expressing said population of stochastic polynucleotide sequences to produce said population of polynucleotides.

51. The method of claim 47 or 48, wherein said population of polynucleotides comprise DNA.

52. The method of claim 47 or 48, wherein said population of polynucleotides comprise RNA.

53. The method of claim 47, further comprising determining the sequence of said one or more isolated polynucleotide sequences having said predetermined property.

54. The method of claim 47, wherein said predetermined property comprises binding or chemical catalysis.

55. The method of claim 54, further comprising improving said predetermined property by in vitro or in vivo mutagenesis.

56. The method of claim 54, wherein said binding further comprises modification of a biological or chemical property of a compound bound by said polynucleotide.

57. The method of claim 56, wherein said modification of said biological or chemical property of said compound further comprises stimulating or inhibiting at least one biological function of said compound.

58. The method of claim 47, wherein said population of polynucleotides comprises greater than about 1×

10⁶ different stochastic polynucleotide sequences.

59. The method of claim 47, wherein said population of polynucleotides comprises greater than about 1×

10⁷ different stochastic polynucleotide sequences.

60. The method of claim 47, wherein said population of polynucleotides comprises greater than about 1×

10⁸ different stochastic polynucleotide sequences.

61. A method of isolating a polynucleotide having a binding property to a ligand, comprising:

(a) synthesizing a population of stochastic polynucleotide sequences;

(b) inserting said population of stochastic polynucleotide sequences into a population of vectors to form a population of vectors containing stochastic polynucleotide sequences;

(c) expressing in host cells said population of vectors containing stochastic polynucleotide sequences to produce a diverse population of expressed polynucleotides, and

(d) screening said diverse population of polynucleotides with a ligand under conditions which allow binding and detection of one or more polynucleotides to said ligand, and

(e) isolating the stochastic polynucleotide sequence or sequences having said binding property to said ligand.

62. The method of claim 61, wherein said inserting further comprises hybridization of complementary ends.

63. The method of claim 61, wherein said inserting further comprises ligation.

64. The method of claim 61, wherein said population of stochastic polynucleotide sequences are produced by stochastic copolymerization of oligonucleotides or polynucleotides.

65. The method of claim 61, wherein said population of stochastic polynucleotide sequences are produced by copolymerization of nucleotide triphosphates consisting of Adenine, Cytosine, Guanine, Thymine and Uricil.

66. The method of claim 61, wherein said population of stochastic polynucleotide sequences are produced by chemical synthesis.

67. The method of claim 61, wherein said population of stochastic polynucleotide sequences comprise DNA.

68. The method of claim 61, wherein said population of stochastic polynucleotide sequences comprise RNA.

69. The method of claim 61, wherein the vector is a plasmid.

70. The method of claim 61, wherein the vector is viral DNA.

71. The method of claim 61, wherein the vector is a hybrid of plasmid and viral DNA.

72. The method of claim 61, wherein the vector is a phage.

73. The method of claim 61, wherein step (c) further comprises digesting the population of vectors with a restriction enzyme having a recognition sequence absent in the vector and reinserting the digested products into said digested population of vectors to form a different population having a greater number of new stochastic polynucleotide sequences.

74. The method of claim 61, wherein said population of stochastic polynucleotide sequences, comprise at least 1×

10⁴ different polynucleotide sequences.

75. The method of claim 61, wherein said population of stochastic polynucleotide sequences, comprise greater than 1×

10⁵ different polynucleotide sequences.

76. The method of claim 61, wherein said population of stochastic polynucleotide sequences, comprise greater than 1×

10⁶ different polynucleotide sequences.

77. The method of claim 61, wherein said population of stochastic polynucleotide sequences, comprise greater than 1×

10⁷ different polynucleotide sequences.

78. The method of claim 61, wherein said population of stochastic polynucleotide sequences, comprise greater than 1×

10⁸ different polynucleotide sequences.

79. A method of producing a diverse population of polynucleotides, comprising stochastically copolymerizing a population of polynucleotides so as to produce a new population of polynucleotides containing greater than about 1×

10⁶ different polynucleotide sequences.

80. The method of claim 79, wherein said new population of polynucleotides further comprise greater than about 1×

10⁶ different polynucleotide sequences.

81. The method of claim 79, which said new population of polynucleotides further comprise greater than about 1×

10⁷ different polynucleotide sequences.

82. The method of claim 79, wherein said new population of polynucleotides further comprise greater than about 1×

10⁸ different polynucleotide sequences.

83. The method of claim 79, wherein said stochastic copolymerization is effected by hybridization of complementary sequences.

84. The method of claim 79, wherein said stochastic copolymerization is effected by ligation.

85. The method of claim 79, wherein said population of polynucleotides comprise DNA.

86. The method of claim 79, wherein said population of polynucleotides comprise RNA.

87. The method of claim 79, further comprising introducing said new population of polynucleotides into host cells.

88. The method of claim 79, wherein said population of polynucleotides further comprise stochastic polynucleotide sequences.

89. The method of claim 79, wherein said population of polynucleotides further comprise at least partially stochastic polynucleotide sequences.

90. The method of claim 79, wherein said population of polynucleotides further comprises two or more populations of polynucleotides.

91. A method of producing a diverse population of polynucleotides, comprising:

(a) obtaining one or more populations of polynucleotides;

(b) cleaving the one or more populations of polynucleotides, and

(c) stochastically copolymerizing the one or more populations of cleaved polynucleotides so as to produce a new population of greater than about 1×

10⁵ different polynucleotide sequences.

92. The method of claim 91, wherein said new population of polynucleotides further comprise greater than about 1×

10⁶ different polynucleotide sequences.

93. The method of claim 91, which said new population of polynucleotides further comprise greater than about 1×

10⁷ different polynucleotide sequences.

94. The method of claim 91, wherein said new population of polynucleotides further comprise greater than about 1×

10⁸ different polynucleotide sequences.

95. The method of claim 91, wherein said stochastic copolymerization is effected by hybridization of complementary sequences.

96. The method of claim 91, wherein said stochastic copolymerization is effected by ligation.

97. The method of claim 91, wherein said one or more populations of polynucleotides comprise DNA.

98. The method of claim 91, wherein said one or more populations of polynuclectides comprise RNA.

99. The method of claim 91, further comprising introducing said new population of polynucleotides into host cells.

100. The method of claim 91, wherein said population of polynucleotides further comprise stochastic polynucleotide sequences.

101. The method of claim 91, wherein said population of polynucleotides further comprise at least partially stochastic polynucleotide sequences.

102. The method of claim 91, wherein said population of polynucleotides further comprises two or more populations of polynucleotides.

103. An isolated population of polynucleotides, comprising greater than about 1×

10⁵ different stochastic polynucleotide sequences.

104. The isolated population of claim 103, further comprising greater than about 1×

10⁶ different stochastic polynucleotide sequences.

105. The isolated population of claim 103, further comprising greater than about 1×

10⁷ different stochastic polynucleotide sequences.

106. The isolated population of claim 103, further comprising greater than about 1×

10⁸ different stochastic polynucleotide sequences.

107. The isolated population of claims 103, 104, 105 or 106, wherein said stochastic polynucleotide sequences comprise DNA or RNA.