Method of providing a hybrid polypeptide exhibiting an activity of interest

• US 6,270,968 B1
• Filed: 11/05/1998
• Issued: 08/07/2001
• Est. Priority Date: 05/10/1996
• Status: Expired due to Fees

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novozymes A/S (Novozymes)
• Inventors: Kauppinen, Markus Sakari, Diderichsen, Børge, Sandal, Thomas, Dalbøge, Henrik
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/189,060
• Time in Patent Office: 1,006 Days
• Field of Search: 435/6, 435/91.2, 435/29, 435/69.1, 435/455, 435/471, 435/476
• US Class Current: 435/6.14
• CPC Class Codes: C12N 15/1044 Preparation or screening of libraries displayed on scaffold proteins
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12N 9/248 Xylanases
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01008 Endo-1,4-beta-xylanase (3.2.1.8)
• CPC Class Codes: C12Y 302/01032 Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase

Abstract

A method for providing a hybrid polypeptide having an activity of interest, by i) performing PCR amplification using an uncharacterized DNA sample and oligonucleotide primers with homology to one or more known genes encoding a polypeptide exhibiting said activity of interest, to obtain one or more PCR products, ii) linking the obtained PCR products to a 5′ structural gene sequence and a 3′ structural gene sequence, wherein the 5′ and 3′ structural gene sequences are derived from one or more genes encoding a polypeptide exhibiting said activity of interest, to form hybrid DNA sequences, iii) expressing the resulting hybrid DNA sequences, and iv) screening the hybrid polypeptides to identify a sequence encoding a polypeptide exhibiting said activity of interest or a related activity.

Claims

1. A method for providing a hybrid polypeptide having an activity of interest, comprising:

i) performing PCR amplification using an uncharacterized DNA sample and oligonucleotide primers with homology to one or more known genes encoding a polypeptide exhibiting the activity of interest, to obtain one or more PCR products of unknown sequence;

ii) linking the obtained PCR products of unknown sequence to a 5′

structural gene sequence and a 3′

structural gene sequence, wherein the 5′ and

3′

structural gene sequences are derived from one or more genes encoding a polypeptide exhibiting the activity of interest, to form hybrid DNA sequences;

iii) expressing the hybrid DNA sequences; and

iv) screening the expressed hybrid DNA sequences to identify a sequence encoding a polypeptide exhibiting the activity of interest or a related activity.

2. The method of claim 1, wherein the primers in step i) have homology to conserved regions in one or more known structural genes or encode peptides or polypeptides having homology to known structural genes.

3. The method of claim 2, wherein the PCR primers in step i) are degenerate.

4. The method of claim 1, wherein the uncharacterized DNA sample is naturally occurring DNA.

5. The method of claim 1, wherein the uncharacterized DNA sample is derived from a microorganism which has not been subjected to in vitro selection.

6. The method of claim 1, wherein the uncharacterized DNA sample is derived from a non-isolated microorganism.

7. The method of claim 1, wherein the 5′

structural gene sequence and the 3′

structural gene sequence originate from two different structural genes encoding polypeptides having the same activity.

8. The method of claim 1, wherein the 5′

structural gene sequence and the 3′

structural gene sequence originate from the same structural gene sequence.

9. The method of claim 1, wherein the 5′

structural gene sequence and the 3′

structural gene sequence originate from two different structural gene sequences encoding polypeptides having different activities.

12. The method of claim 1, wherein the DNA sample is derived form a non-cultivable organism.

13. The method of claim 12, wherein the non-cultivable organism is selected from the group consisting of algae, fungi, and protozoa.

14. The method of claim 13, wherein the non-cultivable organism is selected from the group consisting of extremophiles and plantonic marine organisms.

23. The method of claim 1, wherein the activity of interest is an enzymatic activity.

24. The method of claim 23, wherein the enzymatic activity is assayed by wash performance.

25. The method of claim 1, further comprising isolating the hybrid polypeptide exhibiting the activity of interest identified in step iv).

26. The method of claim 25, further comprising sequencing the hybrid DNA sequence encoding the identified hybrid pglypeptide of step iv).

27. The method of claim 1, wherein the hybrid polypeptide identified in step (iv) exhibits an altered activity relative to the activity of the polypeptide encoded by said 5′

and 3′

structural gene sequences.

10. A method for providing a hybrid polypeptide exhibiting an activity of interest, comprising:

i) performing PCR amplification using a DNA sample derived from one or more microorganisms and PCR primers homologous to conserved regions of a known gene encoding a polypeptide exhibiting the activity of interest to obtain one or more PCR products of unknown sequence;

ii) inserting the obtained one or more PCR products of unknown sequence into a gene encoding a parent polypeptide having the activity of interest, wherein the gene is situated in an expression vector, wherein a library or expression vectors containing hybrid DNA sequences is provided;

iii) transforming the expression vector library into a suitable host cell to obtain a library of transformed clones;

iv) culturing the library of step iii) under suitable conditions for expression of said hybrid polypeptides; and

v) screening the library of hybrid polypeptides to identify a polypeptide exhibiting the activity of interest or related activity.

11. The method of claim 10, wherein the DNA sample is derived from a prokaryotic or eukaryotic microorganism.

15. The method of claim 10, wherein the DNA sample is derived from a cultivable organism.

16. The method of claim 15, wherein the cultivable organism is selected from the group consisting of bacteria, filamentous fungi, and yeasts.

17. The method of claim 16, wherein the DNA sample comprises a plasmid.

18. The method of claim 10, wherein the activity of interest is an enzymatic activity.

19. The method of claim 18, wherein the enzymatic activity is selected from the group consisting of phosphatases, oxidoreductases, transferases, and hydrolases.

20. The method of claim 10, wherein the host cell is a microorganism selected from the group consisting of yeast and bacteria.

21. The method of claim 20, wherein the host cell is Saccharomyces cerevisiae.

22. The method of claim 20, wherein the host cell is a bacterium selected from the group consisting of Bacillus sp and Escherchia coli.

28. The method of claim 10, further comprising isolating the hybrid DNA sequence encoding the hybrid polypepide exhibiting the activity of interest identified in step v).

29. The method of claim 10, further comprising determining the sequence of the inserted PCR product.

30. The method of claim 10, wherein the gene into which the PCR product is inserted is not identical to the gene from which the PCR product is obtained.

31. The method of claim 10, wherein the hybrid polypeptide identified in step (v) exhibits an altered activity relative to the activity of the parent polypeptide.

32. A method of providing a hybrid polypeptide exhibiting an activity of interest, comprising:

i) performing PCR amplification using a DNA sample derived from one or more microorganisms using a first primer and a second primer to provide a PCR fragment of an unknown gene having the activity of interest to obtain one or more PCR products of unknown sequence;

wherein the first primer has an exact sequence matching a sequence at the 5′

end of a known gene encoding a polypeptide having the activity of interest and a degenerated consensus sequence;

wherein the second primer has an exact sequence matching a sequence at the 3′

end of a known gene encoding a polypeptide having the activity of interest and a degenerated consensus sequence;

ii) inserting the obtained one or more PCR products of unknown sequence into a gene encoding a parent polypeptide having the activity of interest, wherein the gene is situated in an expression vector, wherein a library or expression vectors containing hybrid DNA sequences is provided;

iii) transforming the expression vector library into a suitable host cell to obtain a library of transformed clones;

iv) culturing the library of step iii) under suitable conditions for expression of said hybrid polypeptides; and

v) screening the library of hybrid polypeptides to identify a polypeptide exhibiting the activity of interest or related activity.

33. The method of claim 32, wherein the first PCR primer comprises an exact sequence of the 5′

end of a known gene encoding a polypeptide having the activity of interest and a sequence comprising a restriction site corresponding to a restriction site in the expression vector; and

wherein the second PCR primer comprises an exact sequence of the 3′

end of a known gene encoding a polypeptide having the activity of interest and a sequence comprising a restriction site corresponding to a restriction site in the expression vector.