Cell-free synthesis and isolation of novel genes and polypeptides

• US 5,658,754 A
• Filed: 11/29/1991
• Issued: 08/19/1997
• Est. Priority Date: 10/05/1989
• Status: Expired due to Term

• Current Assignee: Optein, Inc. (Astrazeneca PLC)
• Original Assignee: Optein, Inc. (Astrazeneca PLC)
• Inventors: Kawasaki, Glenn H.
• Primary Examiner(s): Wax, Robert A.
• Application Number:
  US07/798,985
• Time in Patent Office: 2,090 Days
• Field of Search: 435/68.1, 435/69.1, 435/69.2, 435/69.3, 435/69.4, 435/69.5, 435/69.6, 435/172.3, 435/172.1, 938/3, 938/4, 938/10, 938/11, 938/12, 938/13, 938/14, 938/15, 938/16, 938/19, 938/21, 938/44
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/1041 Ribosome/Polysome display, e.g. SPERT, ARM
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12Q 1/6867 Replicase-based amplification, e.g. using Q-beta replicase

Abstract

A method for the cell-free synthesis and isolation of novel genes and polypeptides is provided. Within one embodiment, an expression unit is constructed onto which semi-random nucleotide sequences are attached. The semi-random nucleotide sequences are first transcribed to produce RNA, and then translated under conditions such that polysomes are produced. Polysomes which bind to a substance of interest are then isolated and disrupted; and the released mRNA is recovered. The mRNA is used to construct cDNA which is expressed to produce novel polypeptides.

Claims

1. A method for producing novel polypeptides, comprising:

(a) constructing an in vitro expression unit comprising a 5'"'"' untranslated region containing an RNA polymerase binding site, a ribosome binding site, and a translation initiation signal, said expression unit being capable of producing mRNA;

(b) attaching one or more semi-random polynucleotides to said expression unit;

(c) transcribing or replicating from the RNA polymerase binding site the polynucleotides associated with the expression unit and semi-random polynucleotides to produce mRNA;

(d) translating said mRNA to produce polysomes under conditions sufficient to maintain said polysomes;

(e) binding said polysomes to a substance of interest;

(f) isolating said polysomes that bind to said substance of interest;

(g) disrupting said isolated polysomes to release mRNA;

(h) recovering said mRNA;

(i) constructing cDNA from said recovered mRNA; and

(j) expressing said cDNA to produce novel polypeptides.

2. The method of claim 1 wherein, after the step of constructing the cDNA, the cDNA is amplified by polymerase chain reaction.

3. The method of claim 1 wherein said semi-random polynucleotide comprises deoxyribonucleic acid.

4. The method of claim 1 wherein said semi-random polynucleotide comprises ribonucleic acid.

5. The method of claim 1 wherein the RNA polymerase binding site is a RNA-directed RNA polymerase binding site.

6. The method of claim 5 wherein said RNA-directed RNA polymerase is Q-Beta replicase.

7. The method of claim 1 wherein, subsequent to the step of recovering, amplifying the mRNA.

8. The method of claim 7 wherein the step of amplifying comprises synthesizing polynucleotide with an RNA-dependent RNA polymerase.

9. The method of claim 8 wherein the RNA-dependent RNA polymerase is Q-Beta replicase.

10. The method of claim 1 wherein the step of isolating comprises removing polysomes that do not bind to said substance of interest by serial dilution or flow-through wash steps.

11. The method of claim 1 wherein, subsequent to the step of isolating said polysomes, said polysomes are exposed to selected stringency conditions such that said polysomes are released from said substance of interest.

12. The method of claim 11 wherein the step of exposing said polysomes comprises raising the temperature, lowering the salt concentration, or raising the metal ion concentration of said polysomes.

16. The method of claims 1, 13 or 14 wherein said ribosome binding site is a prokaryotic ribosome binding site.

17. The method of claims 1, 13 or 14 wherein said ribosome binding site is a viral ribosome binding site.

18. The method of claims 1, 13 or 14 wherein said ribosome binding site is a eukaryotic ribosome binding site.

19. The method of claims 1, 13 or 14 wherein said ribosome binding site comprises the vertebrate consensus translation initiation sequence GCCGCCACCATGG, or functionally equivalent polynucleotides.

20. The method of claims 1, 13 or 14 wherein the expression unit further comprises a polynucleotide which codes for a selected amino-terminal ID peptide, said polynucleotide positioned at the 3'"'"' end of the initiation codon.

21. The method of claim 1, 13 or 14 wherein the expression unit further comprises a 3'"'"' region of a selected polynucleotide, said selected polynucleotide selected from the group consisting of polynucleotides enhancing the amplification, cloning, replication, purification, and isolation of the novel genes.

22. The method of claim 21 wherein the 3'"'"' region includes palindromic polynucleotides to impede ribosome translocation.

23. The method of claim 21 wherein said 3'"'"' region includes a C-terminal polynucleotide which codes for an ID peptide.

24. The method of claim 23 wherein the C-terminal polynucleotide coding for an ID peptide comprises a repetitive sequence.

25. The method of claim 23 wherein the C-terminal polynucleotide codes for a peptide capable of binding to antibodies.

26. The method of claim 1, 13 or 14 wherein the expression unit further comprises restriction sites to allow expression of the novel gene in vivo.

27. The method of claim 26 wherein at least one of said restriction sites comprises the sequence CCATGG, said site positioned at the start of translation.

28. The method of claim 1, 13 or 14 wherein the 5'"'"' untranslated region of said expression unit includes the promoter for T7, T3, or SP6 polymerase in the 5'"'"' untranslated region.

29. The method of claim 1, 13 or 14 wherein the semi-random polynucleotides are generated by mechanically, chemically, or enzymatically fragmenting naturally-occurring DNA or cDNA.

30. The method of claim 1, 13 or 14 wherein the semi-random polynucleotides are generated by chemically synthesizing polynucleotides to form gene sequences.

31. The method of claim 30 wherein the step of synthetically synthesizing said nucleotides comprises the steps of (1) utilizing substantially equal molar amounts of C, A, and G, and only half of said substantially equal molar amount of T in the first codon positions;

(2)utilizing substantially equal molar amounts of C, T, and G, and only half of said substantially equal molar amount of A in the second codon positions; and

(3) utilizing substantially equal molar amounts of only C and G in the third codon positions.

32. The method of claim 1, 13 or 14 wherein the step of attaching further comprises polymerizing said nucleotides directly onto the 3'"'"' end of the 5'"'"' untranslated region of the expression unit.

33. The method of claim 1 or 13 wherein the step of transcribing comprises transcribing said polynucleotides in the presence of diguanosine triphosphate or analogs thereof.

34. The method of claim 1, 13 or 14 wherein the step of translating comprises translating said polynucleotides in the presence of diguanosine triphosphate or analogs thereof and guanylytransferase.

35. The method of claim 1, 13 or 14 wherein the step of translating is conducted in the presence of nonsense-suppressing tRNAs.

36. The method of claim 35 wherein the nonsense-suppressing tRNA is a tyrosine-inserting, nonsense-suppressing tRNA.

37. The method of claim 1, 13 or 14 wherein said substance of interest is selected from a group consisting of surface antigens, receptor proteins, toxins, organic polymers, metabolites, active sites of protein molecules, hormones, antibodies, and pollutants.

38. The method of claim 1, 13 or 14 wherein said substance of interest is the variable/hypervariable region of an antibody.

39. The method of claim 1, 13 or 14 wherein said substance of interest is a receptor protein.

40. The method of claim 39 wherein said receptor protein is a growth factor receptor protein.

41. The method of claim 40 wherein said growth factor receptor protein is selected from the group consisting of insulin and epidermal growth factor.

42. The method of claim 1, 13 or 14 wherein said substance of interest is selected from the group consisting of viral surface antigen, viral receptor protein and CD4.

43. The method of claim 1, 13 or 14 wherein the step of expressing cDNA comprises chemically synthesizing the peptide encoded by said cDNA.

44. The method of claim 1, 13 or 14 wherein the step of expressing cDNA comprises cloning the polynucleotide into an expression vector for polypeptide synthesis in genetically engineered microorganisms.

45. The method of claim 1, 13 or 14 wherein the step of expressing cDNA comprises in vitro transcription and/or translation of the polynucleotide.

46. The method of claim 1, 13 or 14 wherein the step of expressing cDNA comprises synthesizing a polynucleotide encoding a polypeptide substantially homologous to that encoded by said cDNA, the polypeptide encoded by said polynucleotide being substantially identical to the binding region of said polysomes that bind to the substance of interest.

47. The method of claim 1, 13 or 14 wherein said cDNA is joined to other selected polynucleotides selected from the group consisting of polynucleotides encoding toxins, antibodies, enzymes, biologically active peptides, and peptides capable of binding to antibodies.

13. A method for producing novel polypeptides, comprising:

(a) constructing an in vitro expression unit comprising a 5'"'"' untranslated region containing an RNA polymerase binding site, a ribosome binding site, and a translation initiation signal, said expression unit being capable of producing mRNA;

(b) attaching one or more semi-random polynucleotides to the expression unit;

(c) transcribing from the RNA polymerase binding site the polynucleotides associated with the expression unit and semi-random polynucleotides to produce mRNA;

(d) translating said mRNA in vitro to produce biologically active polypeptides;

(e) subdividing the mRNA encoding said biologically active polypeptides;

(f) transcribing, translating, and subdividing as set forth in steps (c)-(e), such that the gene of interest is isolated;

(g) constructing cDNA from said isolated gene; and

(h) expressing said cDNA to produce novel polypeptides.

15. The method of claim 13 wherein, subsequent to the step of subdividing the RNA, amplifying the novel polynucleotides encoding the biologically active polypeptides with polymerase chain reaction or with an RNA-directed RNA polymerase.

14. A method for producing novel polypeptides, comprising:

(a) constructing an in vitro expression unit comprising a 5'"'"' untranslated region containing an RNA polymerase binding site, a ribosome binding site, and a translation initiation signal, said expression unit being capable of producing mRNA;

(b) attaching one or more semi-random polynucleotides to the expression unit;

(c) replicating from the RNA polymerase binding site the polynucleotides associated with the expression unit and semi-random polynucleotides to produce mRNA;

(d) translating said mRNA in vitro to produce biologically active polypeptides;

(e) subdividing the mRNA encoding said biologically active polypeptides;

(f) translating and subdividing as set forth in steps (d)-(e) such that the gene of interest is isolated;

(g) constructing cDNA from said isolated gene; and

(h) expressing said cDNA to produce novel polypeptides.

48. A method for producing novel polypeptides, comprising:

(a) constructing an in vitro expression unit comprising a 5'"'"' untranslated region containing an RNA polymerase binding site, a ribosome binding site, and a translation initiation signal, said expression unit being capable of producing mRNA;

(b) attaching one or more semi-random polynucleotides to said expression unit adjacent to a polynucleotide sequence encoding a fixed amino acid sequence;

(c) transcribing or replicating from the RNA polymerase binding site the polynucleotides associated with the expression unit and semi-random polynucleotides to produce mRNA which encodes a polypeptide of at least about fifty amino acids;

(d) translating said mRNA to produce polysomes under conditions sufficient to maintain said polysomes;

(e) binding said polysomes to a substance of interest;

(f) isolating said polysomes that bind to said substance of interest;

(g) disrupting said isolated polysomes to release mRNA;

(h) recovering said mRNA;

(i) constructing cDNA from said recovered mRNA; and

(j) expressing said cDNA to produce novel polypeptides.

49. The method of claim 48, wherein the semi-random polynucleotides encode a peptide of about 30 amino acids.

50. The method of claim 48, wherein the polynucleotide sequence encoding a fixed amino acid sequence encodes an amino acid sequence of at least 20 amino acids.