Screening methods in eucaryotic cells

• US 5,989,814 A
• Filed: 04/01/1997
• Issued: 11/23/1999
• Est. Priority Date: 04/01/1997
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Tan, Ruoying, Frankel, Allan
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/847,176
• Time in Patent Office: 966 Days
• Field of Search: 435/4, 435/6, 435/172.2, 435/172.3, 435/240, 435/70, 435/7.31, 435/697, 435/320.1, 435/254.21, 536/23.1, 536/24.3, 536/23.4, 536/24.1
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92

Abstract

The invention provides new methods for screening libraries of peptides and other compounds for a desired property in eucaryotic cells. The methods are premised, in part, on the unexpected observation that the contents of procaryotic or lower eucaryotic cells, such as yeast, can be transferred to recipient eucaryotic cells in an essentially clonal manner by protoplast fusion of the respective cells. Applications of the methods include screening peptides in eucaryotic cells substantially incapable of episomal replication of transferred nucleic acid fragments; screening in eucaryotic cells peptides or secondary metabolites produced in procaryotic cells; and screening a library of peptides for capacity to bind a selected RNA in eucaryotic cells.

Claims

1. A method of screening a library of nucleic acid fragments in eucaryotic cells, comprising:

(1) transforming the library of nucleic acid fragments into primary cells, wherein the cells are procaryotic or fungi;

(2) culturing the primary cells under conditions whereby the copy number of nucleic acid fragments is amplified to an average of at least 200 copies per transformed cell;

(3) contacting the transformed primary cells with a population of eucaryotic cells, which lack capacity for episomal replication of the transferred nucleic acid fragments, under conditions whereby outersurfaces of the transformed primary cells and eucaryotic cells fuse and contents of the transformed primary cells including at least some of the library of nucleic acid fragments are transferred to the eucaryotic cells;

(4) screening the nucleic acid fragments in the eucaryotic cells to isolate one or more eucaryotic cells having a desired property conferred by one or more members of the library of nucleic acid fragments, an expression product thereof, or a secondary metabolite of an expression product;

(5) lysing the one or more eucaryotic cells to release the one or more members of the library of nucleic acid fragments and electroporating the nucleic acid fragments into further procaryotic cells;

(6) propagating the further procaryotic cells to amplify the one or more members of the library of nucleic acid fragments, which confer the desired property.

2. The method of claim 1, wherein the primary cells are E. coli, the nucleic acid fragments are contained in a ColE1 vector, and the primary cells are cultured in the presence of an antibiotic to amplify the copy number of the library of nucleic acid fragments.

3. The method of claim 1, further comprising isolating a nucleic acid fragment from the amplified further procaryotic cells, which confers the desired property.

4. The method of claim 1, further comprising repeating steps (2)-(5), wherein the amplified further procaryotic cells in step (5) of a previous cycle form the transformed primary cells in step (2) of the next cycle.

5. The method of claim 1, wherein the library of nucleic acid fragments has at least 10⁵ members.

6. The method of claim 1, wherein at least ten different members of the library of nucleic acid fragments are electroporated from the one or more eucaryotic cells to the further procaryotic cells.

7. The method of claim 1, wherein the library of nucleic acid fragments are expressed in the eucaryotic cells before the screening.

8. The method of claim 1, wherein the nucleic acid fragments encode different peptides, and one or more of the peptides confers the desired property in the eucaryotic cells.

9. The method of claim 1, wherein the nucleic acid fragments encode enzymes, which produce secondary metabolites in the procaryotic cells, which are transferred together with at least some of the nucleic acid fragments to the eucaryotic cells, and one or more of the secondary metabolites confers the desired property in the eucaryotic cells.

10. The method of claim 8, wherein, in the screening step, the eucaryotic cells contain a construct encoding a reporter enzyme operably linked to a regulatory sequence, and the one or more peptides confers the desired property by binding to the regulatory sequence or a transcript thereof inducing expression of the reporter enzyme.

11. The method of claim 10, wherein the transformed primary cells comprise the construct which is transferred into the eucaryotic cells with the library of nucleic acid fragments.

12. The method of claim 10, wherein the reporter is GFP.

13. The method of claim 11, wherein a transcript of the regulatory sequence is recognized by a site-specific RNA binding peptide and the nucleic acid fragments encode the RNA binding peptide.

14. The method of claim 13, wherein the nucleic acid fragments encode fusion proteins comprising an RNA binding peptide and a transcriptional inducer, the RNA binding peptide differing among fusion proteins.

15. The method of claim 14 wherein the construct encodes GFP operably linked to a promoter induced by HIV-1 TAT and an RNA binding site and the transcriptional inducer is a HIV-1 TAT polypeptide.

16. The method of claim 3, further comprising determining the sequence of the isolated member of the nucleic acid fragment library.

17. The method of claim 16, further comprising producing the expression product of the isolated member of the nucleic acid fragment library.

18. The method of claim 17, further comprising formulating the expression product as a therapeutic composition.

19. The method of claim 1, wherein the nucleic acid fragments are members of a natural library.

20. The method of claim 1, wherein the nucleic acid fragments are members of a randomized library.

21. A method of screening a library of nucleic acid fragments in eucaryotic cells, comprising:

(1) transforming the library of nucleic acid fragments into primary cells, wherein the cells are procaryotic or fungi;

(2) contacting the transformed primary cells with a population of eucaryotic cells under conditions whereby outersurfaces of the transformed primary cells and eucaryotic cells fuse and contents of the transformed primary cells including at least some of the library of nucleic acid fragments are transferred to the eucaryotic cells;

(3) screening the nucleic acid fragments in the eucaryotic cells without substantial replication of the nucleic acid fragments in the eucaryotic cells to isolate one or more eucaryotic cells having a desired property conferred by one or more members of the library of nucleic acid fragments, an expression product thereof, or a secondary metabolite of an expression product;

(4) transferring the one or more members of the library of nucleic acid fragments into further procaryotic cells;

(6) propagating the further procaryotic cells to amplify the one or more members of the library of nucleic acid fragments, which confer the desired property.

33. The method of claim 21, wherein the nucleic acid fragments are not expressed in the eucaryotic cells.

22. A method of screening a library of nucleic acid fragments in eucaryotic cells, comprising:

(1) transforming the library of nucleic acid fragments into cells, wherein the cells are procaryotic cells or fungi, and culturing the cells under conditions in which expression products and/or secondary metabolites of expression products are produced;

(2) contacting the transformed cells with a population of eucaryotic cells under conditions whereby outersurfaces of the transformed procaryotic and eucaryotic cells fuse and contents of the transformed procaryotic cells including at least some of the library of nucleic acid fragments, expression products thereof and/or secondary metabolites of expression products are transferred to the eucaryotic cells, whereby at least some eucaryotic cells receive an expression product and/or a secondary metabolite thereof and a nucleic acid fragment encoding the expression product;

(3) screening the eucaryotic cells to isolate one or more eucaryotic cells having a desired property conferred by one or more of the expression products or one or more of the secondary metabolites produced in the primary cells;

(4) transforming the one or more member of the library of nucleic acid fragments from the one or more eucaryotic cells into further procaryotic cells;

(5) propagating the transformed further procaryotic cells to amplify the one or more members of the library of nucleic acid fragments, which produce the one or more expression products and/or one or more secondary metabolites that confer the desired property.

23. A method of screening for RNA binding peptides in a eucaryotic cells, comprising:

introducing into a population of eucaryotic cells a library of nucleic acid fragments encoding fusion proteins;

a fusion protein comprising a peptide linked to a transcriptional inducer, the peptides varying between fusion proteins;

wherein the eucaryotic cells further comprise a construct encoding a reporter gene operably linked to a promoter from which expression is stimulated by the transcriptional inducer and an RNA binding site;

whereby one or more fusion proteins, each comprising a peptide having specific affinity for the RNA binding site bind to the RNA binding site of the reporter construct or a transcript thereof via the peptide, and the transcriptional inducer linked to the peptide stimulates expression of the reporter gene from the promoter;

isolating one or more eucaryotic cells with stimulated expression of the reporter gene, the one or more cells containing one or more nucleic acid fragments encoding the one or more fusion proteins comprising a peptide having specific affinity for the RNA binding site wherein the introducing step comprises;

(1) transforming the library of nucleic acid fragments into primary cells, which are procaryotic cells or fungi;

(2) contacting the transformed primary cells with the population of eucaryotic cells under conditions whereby outersurfaces of the transformed primary and eucaryotic cells fuse and contents of the transformed primary cells including at least some of the library of nucleic acid fragments are transferred to the eucaryotic cells; and

the method further comprises

(3) transforming nucleic acid fragments from the isolated one or more eucaryotic cells into further procaryotic cells;

(4) propagating the transformed further procaryotic cells to amplify one or more members of the library of nucleic acid fragments encoding fusion proteins comprising a peptide with a specific affinity for the RNA binding site.

24. The method of claim 23, wherein the primary cells are E. coli, the nucleic acid fragments are contained in a ColE1 vector, and the primary cells are cultured in the presence of an antibiotic to amplify the copy number of the library of nucleic acid fragments.

25. The method of claim 24, wherein the eucaryotic cells lack capacity for episomal replication of the transferred nucleic acid fragments.

26. The method of claim 23, wherein the transcriptional inducer is a HIV TAT polypeptide and the promoter is a HIV LTR promoter.

27. The method of claim 26, wherein the RNA binding site is a HIV RRE site.

28. The method of claim 27, wherein the HIV TAT polypeptide lacks a natural HIV Tat RNA binding domain.

29. The method of claim 23, wherein the construct is transferred to the eucaryotic cells as a component of the contents of the primary cells.

30. The method of claim 23, further comprising formulating synthesizing the peptide having specific affinity for the RNA binding site.

31. The method of claim 23, further comprising formulating the peptide having specific affinity for the RNA binding site in a therapeutic or diagnostic composition.

32. A method of screening RNA sequences for specific affinity to a selected peptide in eucaryotic cells, comprising:

introducing into a population of eucaryotic cells a library of variant forms of a first construct, the first construct encoding a reporter gene operably linked to a promoter from which expression is stimulated by a transcriptional inducer and a potential RNA binding site, which varies between the variant forms;

wherein the eucaryotic cells further comprise a second construct encoding a fusion protein comprising a transcriptional inducer linked to the selected peptide;

whereby the fusion protein binds to one or more variant forms of the first construct or transcripts thereof, each having a potential RNA binding site with specific affinity for the peptide, stimulating expression of the reporter gene from the promoter;

isolating one or more eucaryotic cells with stimulated expression of the reporter gene, the one or more cells containing one or more variant forms of the first construct encoding one or more potential RNA binding sites with specific affinity for the selected peptide.