Optimization of crossover points for directed evolution
AssigneeMaxygen, Inc.
InventorsEmily C. Mundorff, Sridhar Govindarajan, Claes Gustafsson, Jeremy S. Minshull
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Abstract
Methods and devices for more efficiently engineering diversity into recombinant polypeptides and/or nucleic acids are provided herein. For example, a variety of methods of selecting and/or assessing potential crossover sites in an amino acid sequence or a nucleotide sequence are provided, as well as the resulting chimeric product sequences. These methods include, e.g., consideration of structural, functional and/or statistical data in the selection and assessment of sequences and crossover sites for use in recombination.
Claims63
A method of determining the fitness of multiple potential crossover points on a reference peptide sequence, the method comprising:
- (a) for each of the multiple potential crossover points on the reference peptide sequence, calculating an overall value of a fitness parameter from multiple individual values of the fitness parameter for multiple chimeras having the potential crossover point under consideration;
- and
- (b) based on the respective overall values of the fitness parameter for the potential crossover points, choosing an actual crossover point for a chimeric peptide comprising a partial sequence of the reference peptide sequence.
A computer program product comprising a machine readable medium on which is provided program instructions for determining the fitness of multiple potential crossover points on a reference peptide sequence, the program instructions comprising:
- (a) code for calculating, for each of the multiple potential crossover points on the reference peptide sequence, an overall value of a fitness parameter from multiple individual values of the fitness parameter for multiple chimeras having the potential crossover point under consideration;
- and
- (b) code for choosing, based on the respective overall values of the fitness parameter for the potential crossover points, an actual crossover point for a chimeric peptide comprising a partial sequence of the reference peptide sequence.
A computer-implemented method for determining the fitness of two or more potential crossover points, the method comprising:
- (a) identifying a first potential crossover point in a reference peptide sequence;
- (b) generating a first chimeric sequence having the potential crossover point with and comprising one partial sequence from the reference peptide sequence and another partial sequence from a different sequence;
- (c) applying the first chimeric sequence to a contact map for the reference peptide sequence;
- (d) calculating a value of a fitness parameter from residue-residue interactions in the first chimeric sequence selected using the contact map;
- (e) repeating (b)-(d) for one or more additional chimeric sequences;
- (f) calculating an overall fitness value from the value of the fitness parameter for each chimeric sequence considered in (b)-(e);
- (g) identifying a second potential crossover point in the reference peptide sequence;
- and
- (h) performing (b)-(f) for the second potential crossover point.
A method of selecting crossover points between two or more biomolecules, the method comprising:
- i) providing a reference sequence of a reference biomolecule;
- ii) generating a contact map for the reference sequence;
- iii) providing a first sequence of a first biomolecule and a second sequence of a second biomolecule, between which one or more crossover points are determined;
- iv) aligning the first and second sequences with the reference sequence;
- v) replacing a subsequence from the first sequence with a subsequence from the second sequence to produce a chimeric biomolecule sequence, wherein the subsequences terminate at a selected crossover point,;
- vi) comparing the chimeric biomolecule sequence with the contact map to select two or more elements in the chimeric biomolecule sequence that correspond to proximal elements in the contact map of the reference biomolecule;
- and
- vii) scoring the selected elements, wherein the score provides a measure of the likelihood of the chimeric biomolecule sequence having a property similar or identical to the reference biomolecule.
A computer-readable medium comprising computer code that
- i) inputs a reference sequence of a reference biomolecule.
- ii) generates a contact map for the reference sequence;
- iii) aligns a first sequence and a second sequence with the reference sequence;
- iv) replacing a subsequence comprising first and second crossover sites on the first sequence with a corresponding subsequence from the second sequences to produce a chimeric sequence;
- v) compares the chimeric sequence with the contact map to select two or more elements in the chimeric amino acid sequence that correspond to proximal elements in the contact map;
- and
- vi) scores the selected elements.
Dependencies are inferred from the claim text.