Cell-free synthesis and isolation of novel genes and polypeptides

• US 5,643,768 A
• Filed: 06/06/1995
• Issued: 07/01/1997
• 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.
• Assistant Examiner(s): BUGAISKY, GABRIELE E
• Application Number:
  US08/469,026
• Time in Patent Office: 756 Days
• Field of Search: 536/23.1, 536/23.2, 435/68.1, 435/69.1, 435/69.2, 435/69.3, 435/69.4, 435/69.5, 435/69.6, 435/172.1, 435/172.3, 435/91.21, 435/91.51, 435/91.3, 435/91.1, 435/91.2 , 435/91.5, 935/3, 935/4, 935/10, 935/16, 935/17, 935/19, 935/20, 935/21
• US Class Current: 506/10
• 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 isolating a nucleotide sequence which encodes a polypeptide of interest, comprising

transcribing an expression unit which comprises a 5'"'"' untranslated region containing an RNA polymerase binding sequence, a ribosome binding sequence, a translation initiation signal, and one or more semi-random nucleotide sequences to produce a mRNA library;

translating in vitro the mRNA library under conditions which maintain polysomes having polypeptide chains attached thereto;

contacting the polysomes to a substance of interest and isolating mRNA from the polysomes that specifically bind to the substance of interest.

2. The method according to claim 1, wherein subsequent to isolating mRNA from the polysomes that specifically bind to the substance of interest, constructing cDNA from said recovered mRNA and expressing said cDNA.

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

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

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

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

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

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

9. The method of claim 1 wherein the step of isolating comprises removing polysomes that do not bind to said substance of interest.

10. The method of claim 1 wherein the semi-random nucleotide sequence encodes a peptide of at least 30 amino acids.

11. The method of claim 1 wherein the one or more semi-random polynucleotides is attached to said expression unit adjacent to a polynucleotide sequence encoding a predetermined amino acid sequence.

12. The method of claim 11 wherein the expression unit encodes a peptide of at least 50 amino acids.

13. The method of claim 12, wherein the semi-random polynucleotides encode a peptide of about 30 amino acids.

14. The method of claim 12, wherein the polynucleotide sequence encoding a fixed amino acid sequence encodes an amino acid sequence of at least 20 amino acids.

15. A method for isolating a nucleotide sequence which encodes a polypeptide of interest, comprising:

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

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

(c) transcribing or replicating from the RNA polymerase binding sequence the sequences associated with the expression unit and semi-random nucleotide sequences 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 said mRNA;

(h) recovering said mRNA, thereby isolating a nucleotide sequence which encodes a polypeptide of interest.

16. The method of claim 15 wherein said ribosome binding sequence is a eukaryotic ribosome binding sequence.

17. The method of claim 15 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 nucleotide sequences of interest.

18. The method of claim 17 wherein the 3'"'"' region includes palindromic polynucleotides to impede ribosome translocation.

19. The method of claim 17 wherein said 3'"'"' region includes a C-terminal polynucleotide which codes for an identification peptide.

20. The method of claim 19 wherein the C-terminal polynucleotide codes for a peptide capable of binding to antibodies.

21. The method of claim 15 wherein the 5'"'"' untranslated region of said expression unit includes the promoter for T7, T3, or SP6 polymerase in the 5'"'"' untranslated region.

22. The method of claim 15 wherein the semi-random polynucleotides are generated by mechanically, chemically, or enzymatically fragmenting naturally-occurring DNA or cDNA.

23. The method of claim 15 wherein the semi-random polynucleotides are generated by chemically synthesizing polynucleotides to form gene sequences.

24. The method of claim 23 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.

25. The method of claim 15 wherein the step of attaching further comprises polymerizing said nucleotides directly onto the 3'"'"' end of the 5'"'"' untranslated region of the expression unit.

26. The method of claim 15 wherein the step of translating is conducted in the presence of nonsense-suppressing tRNAs.

27. The method of claim 15 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.

28. The method of claim 15 wherein said substance of interest is a receptor protein or a variable/hypervariable region of an antibody.

29. A method for isolating a nucleotide sequence which encodes a polypeptide of interest, 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; and

(e) contacting said polysomes to a substance of interest and isolating mRNA from the polysomes that specifically react with the substance of interest.

30. The method of claim 29, wherein the semi-random polynucleotides encode a peptide of about 30 amino acids.

31. The method of claim 29, wherein the polynucleotide sequence encoding a fixed amino acid sequence encodes an amino acid sequence of at least 20 amino acids.