Method for the construction of randomized gene sequence libraries in cells

1. A method for the construction of randomized gene libraries in yeast cells sensitive to Kluyveromyces lactis γ toxin and which are capable of homologous recombination comprising the following steps:

introducing into said yeast cells

a) a target vector comprising a first DNA sequence coding for at least a γ-subunit of a Kluyveromyces lactis killer toxin as negative selection marker, wherein said Kluyveromyces lactis killer toxin lacks the signal peptide and wherein said DNA sequence is flanked at its 5′ end by a first target sequence and at its 3′ end by a second target sequence and;

b) a donor DNA sequence which is flanked at its 5′ end by a DNA sequence which is homologous to said first target sequence and flanked at its 3′ end by a DNA sequence which is homologous to said second target sequence; and

cultivation of said yeast cells under conditions allowing the selection of cells in which said DNA sequence in the target vector encoding at least a γ-subunit of a Kluyveromyces lactis killer toxin has been replaced by said donor sequence by means of homologous recombination thereby abolishing expression of said γ-subunit of a K. lactis killer toxin.

2. The method of claim 1 , wherein said target vector further comprises a second DNA sequence encoding at least one protein region.

3. The method of claim 2 wherein said first DNA sequence of said target vector flanked by said two target sequences is present within a protein region encoding DNA sequence of said second DNA sequence comprised in said target vector.

4. The method of claim 1 wherein said DNA sequence encoding at least the γ subunit of the K. lactis killer toxin is under control of a heterologous promoter.

5. The method of claim 4 wherein said promoter is located between the DNA sequence encoding at least the γ subunit of K. lactis killer toxin and one of the two target sequences.

6. The method of claim 1 , wherein said first DNA sequence of said target vector comprises at least one restriction enzyme recognition site that is unique for a given restriction enzyme.

7. The method of claim 6 , wherein said unique recognition site is located in the coding region of the γ-toxin DNA sequence.

8. The method of claim 2 wherein said second DNA sequence encodes an antibody or a single chain antibody.

9. The method of claim 3 wherein said second DNA sequence encodes an antibody or a single chain antibody and wherein said first DNA sequence of said target vector present within at least one CDR encoding region of said antibody or said single chain antibody.

10. The method of claim 8 or 9 wherein said first DNA sequence comprising the γ subunit of K. lactis killer toxin is transcribed in the opposite direction of said antibody or single chain antibody gene.

11. The method of claim 1 wherein said target vector is introduced into said yeast cells in linearized form.

12. The method of claim 11 wherein said target vector is linerarized by cutting with a restriction enzyme recognizing in said first DNA sequence of said target vector said at least one restriction enzyme recognition site that is unique for a given restriction enzyme.

13. The method of claim 1 wherein said donor sequence comprises a DNA sequence encoding a protein region.

14. The method of claim 1 wherein said target vector and said donor sequence are introduced into said yeast cells by co-transformation.

15. The method of claim 1 wherein said yeast cells are cultivated at a temperature selected from the range of 24° C. to 30° C.

16. The method of claim 4 wherein the promoter is a constitutive promoter.

17. The method of claim 4 wherein the promoter is a TEF promoter from Ashbya gossypii.

18. The method of claim 6 wherein the restriction enzyme recognition site that is unique for a given restriction enzyme is located between the coding region of the γ-toxin DNA sequence and the promoter.

19. The method of claim 9 wherein the first DNA sequence of said target vector replaces a CDR3VL region of said antibody or said single chain antibody.

20. The method of claim 9 wherein the first DNA sequence of said target vector replaces a CDR2 and a CDR3 region of said antibody or said single chain antibody.

21. The method of claim 1 wherein said yeast cells are Saccharomyces cerevisiae cells.

22. The method of claim 13 wherein said donor sequence comprises a DNA sequence encoding a CDR region of an antibody.

23. The method of claim 2 , wherein said second DNA sequence encodes more than two protein regions.

24. The method of claim 2 wherein said second DNA sequence encodes a full length protein.

25. The method of claim 13 wherein said protein region is a CDR region of an antibody.