Claims
- 1. A method for the mutation, synthesis and selection of a protein of interest, the method comprising:
(a) incubating a replicable RNA molecule encoding the protein with ribonucleoside triphosphate precursors of RNA and an RNA-directed RNA polymerase, wherein the RNA-directed RNA polymerase replicates the RNA molecule but introduces mutations thereby generating a population of mutant RNA molecules; (b) incubating the mutant RNA molecules with a translation system under conditions which result in the synthesis of a population of mutant proteins wherein, after translation, mutant proteins are linked to their encoding RNA molecules; (c) selecting one or more mutant proteins of interest.
- 2. The method as claimed in claim 1, wherein the translation system is a cell-free translation system.
- 3. The method as claimed in claim 1, wherein the mutant proteins are linked to their encoding RNA molecules via ribosome complexes.
- 4. The method as claimed in claim 1, wherein the translation system comprises whole cells.
- 5. The method as claimed in claim 4, wherein the mutant proteins are linked to their encoding RNA molecules by association with or location within the same cell.
- 6. The method as claimed in claim 1, wherein the selecting in step (c) comprises exposing the mutant protein to a target molecule.
- 7. The method as claimed in claim 1, wherein the RNA-directed RNA polymerase
(i) introduces mutations into the replicated RNA molecule at a frequency of at least one point mutation in 104 bases; or (ii) introduces at least one insertion or deletion at a frequency of 10−4.
- 8. The method as claimed in claim 1, wherein the RNA-directed RNA polymerase
(i) introduces mutations into the replicated RNA molecule at a frequency of at least one point mutation in 103 bases; or (ii) introduces at least one insertion or deletion at a frequency of 10−3.
- 9. The method as claimed in claim 1, wherein the RNA-directed RNA polymerase is selected from the group consisting of Qβ replicase, Hepatitis C RNA-directed RNA polymerase, Vesicular Stomatitis Virus RNA-directed RNA polymerase, Turnip yellow mosaic virus replicase and RNA bacteriophage phi 6 RNA-dependent RNA polymerase.
- 10. The method as claimed in claim 1, wherein the RNA-directed RNA polymerase is Qβ replicase.
- 11. A method for producing and selecting a mutant protein of interest, the method comprising:
(a) incubating a replicable RNA molecule encoding the protein with ribonucleoside triphosphate precursors of RNA and an RNA-directed RNA polymerase, wherein the RNA-directed RNA polymerase replicates the RNA molecule but introduces mutations thereby generating a population of mutant RNA molecules; (b) translating the mutant RNA molecules in cells wherein, after translation, each mutant protein is associated with or located within the same cell as its encoding RNA molecule; and (c) selecting a cell comprising a mutant protein of interest.
- 12. The method as claimed in claim 11, wherein the selecting in step (c) comprises exposing the cells to a target molecule.
- 13. The method as claimed in claim 11, which further comprises the step of recovering the mutant RNA molecule or the corresponding DNA molecule encoding the mutant protein of interest from the cell selected in step (c).
- 14. The method as claimed in claim 13, which further comprises repeating steps (a) to (c).
- 15. The method as claimed in claim 11, wherein the RNA-directed RNA polymerase
(i) introduces mutations into the replicated RNA molecule at a frequency of at least one point mutation in 104 bases; or (ii) introduces at least one insertion or deletion at a frequency of 10−4.
- 16. The method as claimed in claim 11, wherein the RNA-directed RNA polymerase
(i) introduces mutations into the replicated RNA molecule at a frequency of at least one point mutation in 103 bases; or (ii) introduces at least one insertion or deletion at a frequency of 10−3.
- 17. The method as claimed in claim 11, wherein the RNA-directed RNA polymerase is selected from the group consisting of Qβ replicase, Hepatitis C RNA-directed RNA polymerase, Vesicular Stomatitis Virus RNA-directed RNA polymerase, Turnip yellow mosaic virus replicase and RNA bacteriophage phi 6 RNA-dependent RNA polymerase.
- 18. The method as claimed in claim 11, wherein the RNA-directed RNA polymerase is Qβ replicase.
- 19. A method for producing and selecting a mutant protein of interest, the method comprising:
(a) transcribing a DNA template to produce a replicable RNA molecule, wherein the DNA template comprises:
(i) an untranslated region comprising a control element that promotes transcription of DNA into RNA and a ribosome binding site; (ii) an open reading frame encoding a protein; and (iii) a stemloop structure situated upstream of the open reading frame; (b) incubating the replicable RNA molecule encoding the protein with ribonucleoside triphosphate precursors of RNA and an RNA-directed RNA polymerase, wherein the RNA-directed RNA polymerase replicates the RNA molecule but introduces mutations, thereby generating a population of mutant RNA molecules; (c) incubating the mutant RNA molecules with a translation system under conditions which result in the synthesis of a population of mutant proteins; (d) selecting one or more mutant proteins of interest.
- 20. The method as claimed in claim 19, wherein the translation system comprises intact cells.
- 21. The method as claimed in claim 19, wherein the selecting in step (d) comprises exposing the cells to a target molecule.
Priority Claims (1)
Number |
Date |
Country |
Kind |
PP3445 |
May 1998 |
AU |
|
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application Ser. No. 09/674,677, filed on Dec. 11, 2000, which is the National Phase of PCT/AU99/00341, filed May 7, 1999, designating the U.S. and published as WO 99/58661, with a claim of priority from Australian application no. PP 3445, filed May 8, 1998.
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
09674677 |
Dec 2000 |
US |
Child |
10408930 |
Apr 2003 |
US |