Methods and compositions for polypeptide engineering
First Claim
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1. A method of identifying one or more evolved polynucleotides with a desired property, the method comprising:
- (a) identifying one or more regions of sequence variance in a population of homologous nucleic acid sequences;
(b) providing a plurality of overlapping nucleic acid subsequences of the homologous nucleic acid sequences, which plurality of overlapping nucleic acid subsequences comprises at least one set of degenerate nucleic acids encoding the sequence variance identified in step (a), wherein the overlapping nucleic acid subsequences are produced by a polymerase chain reaction;
(c) recombining the plurality of overlapping nucleic acid subsequences of step (b) to produce a plurality of recombined nucleic acids; and
, (d) screening or selecting the plurality of recombined nucleic acids to identify one or more evolved polynucleotides with the desired property.
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Abstract
Methods are provided for the evolution of proteins of industrial and pharmaceutical interest, including methods for effecting recombination and selection. Compositions produced by these methods are also disclosed.
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Citations
43 Claims
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1. A method of identifying one or more evolved polynucleotides with a desired property, the method comprising:
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(a) identifying one or more regions of sequence variance in a population of homologous nucleic acid sequences;
(b) providing a plurality of overlapping nucleic acid subsequences of the homologous nucleic acid sequences, which plurality of overlapping nucleic acid subsequences comprises at least one set of degenerate nucleic acids encoding the sequence variance identified in step (a), wherein the overlapping nucleic acid subsequences are produced by a polymerase chain reaction;
(c) recombining the plurality of overlapping nucleic acid subsequences of step (b) to produce a plurality of recombined nucleic acids; and
,(d) screening or selecting the plurality of recombined nucleic acids to identify one or more evolved polynucleotides with the desired property. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43)
(i) introducing the recombined nucleic acids into a cell population;
(ii) expressing the recombined nucleic acids in the cell population to produce expressed polypeptides, and, (iii) selecting or screening one or more members of the cell population or the expressed polypeptides to identify the one or more evolved polynucleotides comprising the desired property.
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26. The plurality of overlapping nucleic acid subsequences provided in step (b) of claim 1.
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27. The method of claim 1, further comprising:
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(e) sequencing the one or more evolved polynucleotides of step (d) to produce one or more evolved polynucleotide sequences; and
,(f) repeating steps (a)-(d) using the one or more evolved polynucleotide sequences of step (e) as at least one source of sequence variation to be included in the degenerate nucleic acids provided in step (b).
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28. The method of claim 1, wherein step (a) comprises:
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(i) aligning the homologous nucleic acid sequences to identify the one or more regions of sequence variance; and
,(ii) segmenting the homologous nucleic acid sequences into the overlapping nucleic acid subsequences.
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29. The method of claim 28, wherein (ii) comprises segmenting the homologous nucleic acid sequences using at least one algorithm.
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30. The method of claim 28, wherein the overlapping nucleic acid subsequences comprise defined segments of the homologous nucleic acid sequences.
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31. The method of claim 1, wherein at least one member of the plurality of overlapping nucleic acid subsequences corresponds to an exon of at least one of the homologous nucleic acid sequences.
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32. The method of claim 31, wherein the exon encodes a structural element of a polypeptide selected from the group consisting of:
- loops, alpha helices, subdomains, whole domains, and hydrophobic cores.
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33. The method of claim 1, wherein the one or more evolved polynucleotides of step (d) are subjected to mutagenesis to provide one or more mutagenized polynucleotides.
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34. The method of claim 33, wherein the mutagenesis comprises recursive sequence recombination.
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35. The method of claim 1, wherein the homologous nucleic acid sequences comprise species variants of one another.
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36. The method of claim 1, wherein step (c) comprises recombining the plurality of overlapping nucleic acid subsequences using a ligase.
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37. The method of claim 1, wherein step (c) comprises recombining the plurality of overlapping nucleic acid subsequences using a polymerase and a ligase.
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38. The method of claim 14, wherein the homologous nucleic acid sequences comprise at least about 70% sequence identity.
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39. The method of claim 38, wherein the homologous nucleic acid sequences comprise at least about 80% sequence identity.
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40. The method of claim 39, wherein the homologous nucleic acid sequences comprise at least about 90% sequence identity.
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41. The method of claim 33, wherein the mutagenesis comprises PCR mutagenesis.
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42. The method of claim 33, wherein the mutagenesis comprises oligonucleotide-directed mutagenesis.
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43. The method of claim 33, wherein the mutagenesis comprises site-directed mutagenesis.
Specification