Process for obtaining DNA, RNA, peptides, polypeptides, or proteins, by recombinant DNA technique

• US 5,976,862 A
• Filed: 06/05/1995
• Issued: 11/02/1999
• Est. Priority Date: 03/30/1985
• Status: Expired due to Term

• Current Assignee: Stuart Kauffman
• Original Assignee: Ixsys, Inc. (Integral Partners LLC)
• Inventors: Ballivet, Marc, Kauffman, Stuart Alan
• Primary Examiner(s): Fox, David T.
• Application Number:
  US08/464,327
• Time in Patent Office: 1,611 Days
• Field of Search: 435/6, 435/7.32, 435/69.1, 435/91.1, 435/71.1, 435/91.4, 435/252.3, 435/471, 435/476, 536/23.1
• US Class Current: 435/252.3
• 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 host cell capable of producing a transcription product or a translation product comprising an expression vector, wherein said expression vector comprises at least one stochastic sequence of polynucleotides comprising the steps of:

producing a library of expression vectors capable of producing a transcription product or a translation product, said expression vectors comprising at least one stochastic sequence of polynucleotides, said expression vectors being produced by the following steps;

providing in an appropriate buffer at least three different sequences of oligonucleotides, said oligonucleotides each comprising at least 7 nucleotide residues;

polymerizing said oligonucleotides in a manner to form a stochastic sequence of polynucleotides;

ligating said stochastic sequence of polynucleotides into a linearized expression vector; and

transforming a competent host cell with said ligated expression vector.

4. A host cell produced in accordance with the process of claim 1, 2, or 3.

5. A process for isolating or screening for a host cell clone capable of producing a transcription or translation product comprising:

producing a transformed host cell according to the process of claim 1, 2 or 3;

culturing said transformed host cell;

screening and/or selecting said transformed host cell in order to isolate a specific transformed clone, wherein said specific transformed clone is a host cell whose transformed stochastic sequence is a template for the synthesis of a transcription product or a translation product; and

isolating said selected or screened host cell clone.

6. The process according to claim 1, 2 or 3 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.

7. The process according to claim 1, 2 or 3 wherein said transcription product comprises a product having a desired property and is selected from the group consisting of a RNA or a DNA.

8. The library of expression vectors according to claim 1, 2 or 3 wherein said library comprises stochastic nucleotide sequences encoding for at least 10,000 peptides, polypeptides or proteins.

2. A process for the production of a host cell capable of producing a transcription product or a translation product comprising an expression vector, wherein said expression vector comprises at least one stochastic sequence of polynucleotides comprising the steps of:

producing a library of expression vectors capable of producing a transcription product or a translation product, said expression vectors comprising at least one stochastic sequence of polynucleotides, said expression vectors being produced by the following steps;

linearizing an expression vector;

reacting said linearized expression vector with terminal transferase enzyme in the presence of desired ratios of deoxynucleotide-triphosphates of guanine, cytosine, thymidine, and adenine to form a stochastic polynucleotide sequence at each 3'"'"' extremity of said linearized vector;

hybridizing said stochastic polynucleotide sequence at a 3'"'"' extremity of said linearized expression vector;

synthesizing a second strand from said 3'"'"' ends of said hybridized expression vector by incubating with polymerase; and

transforming a host cell with said expression vector.

3. A process for the production of a host cell capable of producing a transcription product or a translation product comprising an expression vector, wherein said expression vector comprises at least one stochastic sequence of polynucleotides comprising the steps of:

producing a library of expression vectors capable of producing a transcription product or a translation product, said expression vectors comprising at least one stochastic sequence of polynucleotides;

transforming a host cell with said expression vector;

culturing said transformed host cell;

screening and/or selecting said transformed host cell; and

isolating said selected or screened host cell.

9. A method of producing a diverse population of host cells comprising:

(a) synthesizing a diverse population of stochastically generated polynucleotide sequences comprising greater than about 1×

10⁵ different polynucleotide sequences, said method consisting of stochastic copolymerization of double stranded oligonucleotides, copolymerizaton of the four kinds of nucleotide triphosphates consisting of adenine, cytosine, guaninie and thymine, and chemical synthesis, and

(b) inserting said diverse population of stochastically generated polynucleotide sequences into a population of vectors to form a diverse population of vectors containing stochastically generated polynucleotide sequences; and

(c) inserting said diverse population of vectors into host cells.

10. The method of claim 9, which said diverse population of stochastically generated polynucleotide sequences, further comprise greater than about 1×

10⁶ different polynucleotide sequences.

11. The method of claim 9, which said diverse population of stochastically generated polynucleotide sequences, further comprise greater than about 1×

10⁷ different polynucleotide sequences.

12. The method of claim 9, wherein said diverse population of stochastically generated polynucleotide sequences, further comprise greater than about 1×

10⁸ different polynucleotide sequences.

13. The method of claim 9, wherein said inserting further comprises hybridization of complementary ends.

14. The method of claim 9, wherein said inserting further comprises ligation.

15. The method of claim 9, wherein step (a) further comprises synthesizing a population of at least partially stochastic polynucleotide sequences.

16. The method of claim 9, wherein step (b) further comprises digesting the diverse population of vectors with a restriction enzyme having a recognition sequence absent in the expression vector and reinserting the digested products into said digested population of vectors to form a different population having a greater number of stochastic polynucleotide sequences.

17. A method of producing a diverse population of host cells, comprising stochastically copolymerizing a diverse population of vectors containing double stranded polynucleotides so as to produce a new population of vectors containing greater than about 1×

10⁵ different polynucleotide sequences and inserting said new population of vectors into host cells.

18. The method of claim 17, wherein said new population of vectors further comprise greater than about 1×

10⁶ different polynucleotide sequences.

19. The method of claim 17, which said new population of vectors further comprise greater than about 1×

10⁷ different polynucleotide sequences.

20. The method of claim 17, wherein said new population of vectors further comprise greater than about 1×

10⁸ different polynucleotide sequences.

21. The method of claim 17, wherein said stochastic copolymerization is effected by hybridization of complementary ends.

22. The method of claim 17, wherein said stochastic copolymerization is effected by ligation.

23. The method of claim 17, wherein said double stranded polynucleotides further comprise stochastic polynucleotide sequences.

24. The method of claim 17, wherein said double stranded polynucleotides further comprise at least partially stochastic polynucleotide sequences.

25. The method of claim 17, wherein said diverse population of vectors further comprises two or more diverse populations of vectors.

26. A method of producing a diverse populations of host cells, comprising:

(a) obtaining one or more diverse populations of vectors containing diverse sequences of double stranded polynucleotides;

(b) digesting the one or more diverse populations of vectors with a restriction enzyme, and

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

10⁵ different polynucleotide sequences; and

(d) inserting said new population of polynucleotides into host cells.

27. The method of claim 26, wherein said new population of vectors further comprise greater than about 1×

10⁶ different polynucleotide sequences.

28. The method of claim 26, wherein said new population of vectors further comprise greater than about 1×

10⁷ different polynucleotide sequences.

29. The method of claim 26, wherein said new population of vectors further comprise greater than about 1×

10⁸ different polynucleotide sequences.

30. The method of claim 26, wherein said stochastic copolymerization is effected by hybridization of complementary ends.

31. The method of claim 26, wherein said stochastic copolymerization is effected by ligation.

32. The method of claim 26, wherein said double stranded polynucleotides further comprise stochastic polynucleotide sequences.

33. The method of claim 26, wherein said double stranded polynucleotides further comprise at least partially stochastic polynucleotide sequences.

34. The method of claim 26, wherein said diverse population of vectors further comprises two or more diverse populations of vectors.