Protein variant generation by region shuffling

Patent metadata

Field	Value
Publication number	US-9593326-B2
Application number	US-201213577651-A
Country	US
Kind code	B2
Filing date	Jun 25, 2012
Priority date	Dec 9, 2009
Publication date	Mar 14, 2017
Grant date	Mar 14, 2017

Abstract

Region shuffling methods for efficiently introducing diversity and exploring sequence space are described. Libraries produced directly from these methods contain high fractions of protein variants harboring multiple beneficial mutations. Typically, the methods produce these variants efficiently without the need for sequencing beneficial mutants identified at intermediate stages of the process.

First claim

What is claimed is:

1. A method of introducing diversity into a reference protein sequence, the method comprising:
(a) providing a first plurality of mutant proteins derived from the reference protein sequence, wherein the first plurality of mutant proteins each have one or more established beneficial properties and mutations in a first region of the reference protein sequence but not in substantially any other region of the reference protein sequence;
(b) providing a second plurality of mutant proteins derived from the reference protein sequence, wherein the second plurality of mutant proteins each have one or more established beneficial properties and mutations in a second region of the reference protein sequence but not in substantially any other region of the reference protein sequence;
(c) isolating a first group of nucleic acid segments from nucleic acids encoding the first plurality of mutant proteins, each nucleic acid segment in the first group encoding the first region of the reference protein sequence but not encoding substantially any other region of the reference protein sequence, wherein isolating the first group of nucleic acid segments is performed without first sequencing the first group of nucleic acid segments to identify the mutations in the first region;
(d) isolating a second group of nucleic acid segments from nucleic acids encoding the second plurality of mutant proteins, each nucleic acid segment in the second group encoding the second region of the reference protein sequence but not encoding substantially any of the first region of the reference protein sequence, wherein isolating the second group of nucleic acid segments is performed without first sequencing the second group of nucleic acid segments to identify the mutations in the second region; and
(e) assembling at least the isolated nucleic acid segments from (c) and (d) into full length nucleic acid sequences encoding new mutant proteins.

2. The method of claim 1 , wherein the reference protein sequence is a wild type protein sequence.

3. The method of claim 1 or 2 , further comprising generating the first and second plurality pluralities of mutant proteins by a process comprising introducing point mutations into the reference protein sequence.

4. The method of claim 1 or 2 , further comprising generating the first and second plurality pluralities of mutant proteins by a process comprising performing saturation mutagenesis on the reference protein sequence.

5. The method of claim 1 , wherein the first and second pluralities of mutant proteins contains, collectively, at least about 100 mutations.

6. The method of claim 1 , wherein the first and second pluralities of mutant proteins collectively comprise mutants having beneficial mutations spread across the protein sequence.

7. The method of claim 1 , further comprising grouping individual mutant proteins from the first and second pluralities of mutant proteins based on regions of the reference protein or peptide sequence where mutations occur.

8. The method of claim 1 , wherein the first group of nucleic acid segments comprises at least about 10 distinct nucleic acid segments, each having a distinct sequence.

9. The method of claim 1 , wherein isolating the first group of nucleic acid segments comprises amplifying the nucleic acid segments in the first group in a single amplification reaction.

10. The method of claim 9 , wherein the amplifying is performed under conditions that do not substantially amplify nucleic acid segments encoding any regions other than the first region.

11. The method of claim 9 , wherein isolating the second group of nucleic acid segments comprises amplifying the nucleic acid segments in the second group in a single second amplification reaction.

12. The method of claim 1 , wherein the first group of nucleic acid segments comprises at least about 5 distinct nucleic acid segment sequences.

13. The method of claim 1 , wherein isolating the first group of nucleic acid segments is performed without first identifying any mutation contained in the first group of nucleic acid segments.

14. The method of claim 1 , further comprising isolating a third group of nucleic acid segments from nucleic acids encoding a third plurality of mutant proteins derived from the reference protein sequence, wherein the third plurality of mutant proteins each have one or more established beneficial properties and mutations in a third region of the reference but not in substantially any other region of the reference protein sequence, and wherein each nucleic acid segment in the third group encoding the third region of the reference protein sequence but not encoding substantially any of the first or second regions of the reference protein sequence.

15. The method of claim 1 , further comprising isolating at least three more groups of nucleic acid segments, each encoding a different region of the reference protein sequence.

16. The method of claim 1 , further comprising isolating at least five more groups of nucleic acid segments, each encoding a different region of the reference protein sequence.

17. The method of claim 1 , further comprising, prior to (e) isolating at least two additional groups of nucleic acid segments, each encoding a different region of the reference protein sequence but not encoding substantially any of the other region of the reference protein sequence,
wherein (e) comprises assembling the isolated nucleic acid segments from the at least two additional group in addition to the isolated nucleic acid segments from (c) and (d) into full length nucleic acid sequences encoding new mutant proteins.

18. The method of claim 1 , wherein assembling at least the isolated nucleic acid segments from (c) and (d) is performed without using nucleic acid segments exactly encoding a region of the reference protein sequence.

19. The method of claim 1 , wherein the assembling in (e) is performed using an overlap extension Polymerase Chain Reaction.

20. The method of claim 1 , wherein the assembling in (e) is performed without using primers.

21. The method of claim 1 , wherein the assembling in (e) is performed using homologous recombination in yeast.

22. The method of claim 1 , further comprising (f) identifying one or more recombinant proteins encoded by one or more full length nucleic acid sequences from (e), wherein the one or more recombinant proteins have at least one beneficial property.

23. The method of claim 1 , wherein (c) to (e) are performed without determining sequences of the mutant protein sequences.

24. The method of claim 1 , wherein the nucleic acid segments from (c) used to assemble the full length nucleic acid sequences in (e) are present in non-equimolar amounts during the assembling.

25. The method of claim 24 , wherein the non-equimolar amounts are chosen based on one or more properties of associated mutant proteins harboring mutations encoded by the nucleic acid segments present in non-equimolar amounts.

26. The method of claim 1 , wherein the reference protein is an enzyme.

27. The method according to claim 26 , wherein the enzyme is a cellulase, reductase, transferase, transaminase, or isomerase.

28. The method of claim 1 , further comprising:
assaying and sequencing the new mutant proteins; and
developing a sequence activity model from assay and sequence information for the new mutant proteins.

Assignees

• Clark Louis
• Choudhary Trish
• Codexis Inc

Inventors

• Clark Louis
• Choudhary Trish

Classifications

• C12N15/1034 Primary

  Isolating an individual clone by screening libraries · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N9/0004

  Oxidoreductases (1.) · CPC title

• C12N9/24

  acting on glycosyl compounds (3.2) · CPC title

• C12N15/1027

  by DNA shuffling, e.g. RSR, STEP, RPR · CPC title