Methods for identifying and producing polypeptides

• US 7,957,912 B2
• Filed: 09/10/2009
• Issued: 06/07/2011
• Est. Priority Date: 01/19/1999
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis Mayflower Holdings, LLC (Codexis, Inc.)
• Inventors: Selifonov, Sergey A., del Cardayre, Stephen, Tobin, Matthew, Patten, Phillip A., Giver, Lorraine J., Gustafsson, Claes, Minshull, Jeremy, Stemmer, Willem P. C.
• Primary Examiner(s): Zhou; Shubo (Joe)
• Application Number:
  US12/557,463
• Publication Number:
  US 20100056385A1
• Time in Patent Office: 635 Days
• Field of Search: None
• US Class Current: 702/19
• CPC Class Codes: B01J 2219/007 Simulation or vitual synthesis
• CPC Class Codes: G16B 10/00 ICT specially adapted for evolutionary bioinformatics, e.g. phylogenetic tree construction or analysis
• CPC Class Codes: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
• CPC Class Codes: G16B 15/20 Protein or domain folding
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/30 Detection of binding sites or motifs
• CPC Class Codes: G16B 20/50 Mutagenesis
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: G16B 30/20 Sequence assembly
• CPC Class Codes: G16B 35/00 ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
• CPC Class Codes: G16B 35/20 Screening of libraries
• CPC Class Codes: G16B 40/00 ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
• CPC Class Codes: G16B 40/10 Signal processing, e.g. from mass spectrometry [MS] or from PCR
• CPC Class Codes: G16C 20/60 In silico combinatorial chemistry

Abstract

In silico nucleic acid recombination methods, related integrated systems utilizing genetic operators and libraries made by in silico shuffling methods are provided. One disclosed method involves identifying and producing a polypeptide. The method may involve (a) receiving data identifying sequences of two or more parental polypeptides or parental nucleic acids that encode the polypeptides; (b) selecting one or more cross-over sites on the sequences thereby defining one or more recombinant polypeptides or recombinant nucleic acids; (c) selecting at least one of the recombinant polypeptides or recombinant nucleic acids; (d) recombining one or more oligonucleotides, at least one of which has a sequence matching some or all of that of the recombinant polypeptides or recombinant nucleic acids selected in (c); (e) selecting at least some of the recombined oligonucleotides produced in (d); and (f) producing a polypeptide encoded by the selected nucleic acid.

Claims

1. A method of identifying and producing a polypeptide, the method comprising:

(a) receiving data identifying sequences of two or more parental polypeptides or parental nucleic acids that encode the polypeptides;

(b) selecting one or more cross-over sites on the sequences based on structural or statistical information about the parental polypeptides or polypeptides encoded by the parental nucleic acids thereby defining one or more recombinant polypeptides or recombinant nucleic acids that result from cross-overs between the parental polypeptides or nucleic acids at the one or more cross-over sites;

(c) selecting at least one of the recombinant polypeptides or recombinant nucleic acids;

(d) identifying one or more oligonucleotides for recombination by choosing at least one portion of at least one of the recombinant polypeptides or recombinant nucleic acids selected in (c) which at least one portion identifies the one or more oligonucleotides for recombination;

(e) recombining in vitro or in vivo the one or more oligonucleotides identified in (d), alone or in combination with other oligonucleotides to produce recombined oligonucleotides;

(f) screening at least some of the recombined oligonucleotides produced in (e) to select a selected nucleic acid; and

(g) producing a polypeptide encoded by the selected recombined nucleic acid selected in (f).

2. The method of claim 1, wherein the screening in (f) comprises screening polypeptides encoded by the recombined oligonucleotides.

3. The method of claim 1, further comprising repeating at least two of (a)-(f) using sequences of the recombined oligonucleotides generated in (e) or polypeptides encoded thereby.

4. The method of claim 1, further comprising, prior to (e), synthesizing the one or more oligonucleotides identified in (d).

5. The method of claim 1, wherein the screening in (f) comprises (i) expressing the at least some of the recombined oligonucleotides and (ii) screening polypeptides expressed in (i).

6. The method of claim 1, wherein the polypeptide produced in (f) is an industrial enzyme.

7. The method of claim 1, wherein the polypeptide produced in (f) catalyzes a chemical reaction.

8. The method of claim 7, wherein screening at least some of the recombined oligonucleotides comprises detecting phenotypes associated with catalysis of a chemical reaction.

9. The method of claim 1, wherein the recombining in (e) comprises a gene shuffling procedure.

10. A method of identifying and producing a polypeptide, the method comprising:

(a) receiving data identifying sequences of two or more parental polypeptides or parental nucleic acids that encode the polypeptides;

(b) selecting one or more cross-over sites on the sequences based on structural or statistical information about the parental polypeptides or polypeptides encoded by the parental nucleic acids thereby defining one or more recombinant polypeptides or recombinant nucleic acids that result from cross-overs between the parental polypeptides or nucleic acids at the one or more cross-over sites;

(c) selecting at least one of the recombinant polypeptides or recombinant nucleic acids;

(d) synthesizing a nucleic acid having a sequence corresponding to that of at least one portion of at least one of the recombinant polypeptides or recombinant nucleic acids selected in (c); and

(e) producing a polypeptide encoded by the nucleic acid synthesized in (d).

11. The method of claim 10, further comprising screening the polypeptide produced in (e).

12. The method of claim 11, further comprising repeating (a)-(e) using the sequence of the nucleic acid synthesized in (e) or polypeptides encoded thereby as one of the parental polypeptides or parental nucleic acids that encode the polypeptides.

13. The method of claim 10, wherein (e) comprises (i) expressing the nucleic acid synthesized in (d).

14. The method of claim 10, wherein the polypeptide produced in (e) is an industrial enzyme.

15. The method of claim 10, wherein the polypeptide produced in (e) catalyzes a chemical reaction.

16. A method of identifying and producing a polypeptide, the method comprising:

(a) receiving data identifying sequences of two or more parental polypeptides or parental nucleic acids that encode the polypeptides;

(b) selecting one or more cross-over sites on the sequences thereby defining one or more recombinant polypeptides or recombinant nucleic acids that result from cross-overs between the parental polypeptides or nucleic acids at the one or more cross-over sites;

(c) selecting at least one of the recombinant polypeptides or recombinant nucleic acids;

(d) recombining one or more oligonucleotides, at least one of which has a sequence matching at least a portion of that of a recombinant polypeptide or recombinant nucleic acid selected in (c), to produce recombined oligonucleotides;

(e) selecting at least some of the recombined oligonucleotides produced in (d) to select a selected nucleic acid; and

(f) producing a polypeptide encoded by the selected nucleic acid selected in (e).

17. The method of claim 16, wherein the polypeptide produced in (f) is an industrial enzyme.

18. The method of claim 16, wherein the polypeptide produced in (f) catalyzes a chemical reaction.