Back to results
US 2014/0005057 A1Published application
Protein variant generation by region shuffling
AssigneeCodexis, Inc.
InventorsLouis Clark, Trish Choudhary
Life of this patent
Estimating the term…
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.
Claims40
A method of introducing diversity into a reference protein sequence, the method comprising:
- (a) selecting a plurality of mutant proteins derived from the reference protein sequence;
- (b) isolating a first group of nucleic acid segments, each nucleic acid segment in the first group encoding a first region of the reference protein sequence but not encoding substantially any other region of the reference protein sequence, and each nucleic acid segment in the first group encoding its own distinct mutation, which mutation is found in a mutant protein selected in (a);
- (c) isolating a second group of nucleic acid segments, each nucleic acid segment in the second group encoding a second region of the reference protein sequence but not encoding substantially any of the first region of the reference protein sequence, and each nucleic acid segment in the second group encoding its own distinct mutation, which mutation is found in a mutant protein selected in (a);
- and
- (d) assembling at least the isolated nucleic acid segments from (b) and (c) into full length nucleic acid sequences encoding new mutant proteins,
- wherein (b) is performed without determining sequences of members of the first group of nucleic acid segments.
3 more elements
A method of introducing diversity into a reference protein sequence, the method comprising:
- (a) selecting a plurality of mutant proteins derived from the reference protein sequence;
- (b) amplifying a first group of nucleic acid segments in a single amplification reaction, wherein each nucleic acid segment in the first group encodes a first region of the reference protein sequence, but does not encode substantially any other region of the reference protein sequence, and each nucleic acid segment in the first group encodes its own distinct mutation, which mutation is found in a mutant protein selected in (a);
- (c) amplifying a second group of nucleic acid segments in a second amplification reaction, wherein each nucleic acid segment in the second group encodes a second region of the reference protein sequence, but does not encode substantially any of the first region of the reference protein sequence, and each nucleic acid segment in the second group encodes its own distinct mutation, which mutation is found in a mutant protein selected in (a);
- and
- (d) assembling at least the isolated nucleic acid segments from (b) and (c) into full length nucleic acid sequences encoding new mutant proteins.
2 more elements
(canceled)
A method of introducing diversity into a reference protein sequence, the method comprising:
- (a) selecting a plurality of mutant proteins derived from the reference protein sequence;
- (b) amplifying a first group of nucleic acid segments, wherein each nucleic acid segment in the first group encodes a first region of the reference protein sequence, but does not encode substantially any other region of the reference protein sequence, and each nucleic acid segment in the first group encodes its own distinct mutation, which mutation is found in a mutant protein selected in (a);
- (c) fragmenting and optionally recombining the amplified nucleic acids produced in (b) to thereby produce a pool of nucleic acids encoding the first region and having increased diversity;
- (d) amplifying a second group of nucleic acid segments, wherein each nucleic acid segment in the second group encodes a second region of the reference protein sequence, but does not encode substantially any of the first region of the reference protein sequence, and each nucleic acid segment in the second group encodes its own distinct mutation, which mutation is found in a mutant protein selected in (a);
- and
- (e) assembling at least the isolated nucleic acid segments from (c) and (d) into full length nucleic acid sequences encoding new mutant proteins.
3 more elements
Dependencies are inferred from the claim text.