Methods and compositions for polypeptide engineering
First Claim
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1. A method of screening or selecting a further evolved polypeptide for a desired property, the method comprising:
- (a) providing a mixture of nucleic acid subsequences of two or more parental nucleic acid substrate molecules encoding homologous polypeptides, wherein each parental nucleic acid substrate molecule differs from at least one other parental nucleic acid substrate molecule in at least one nucleotide;
(b) ligating two or more of the nucleic acid subsequences together in an ordered reassembly to produce a library of recombinant nucleic acid substrate molecules encoding varients of the homologous polypeptides, which library comprises a plurality of nucleic acid molecules, each member of the plurality comprising at least one subsequence from at least two of the two or more parental nucleic acid substrate molecules, wherein the plurality of nucleic acid molecules are homologous to one another and to the parental substrate nucleic acid molecules;
(c) expressing the library of recombinant nucleic acid substrate molecules to provide evolved polypeptides;
(d) screening or selecting the evolved polypeptides to identify at least one evolved polypeptide with the desired property;
(e) recovering at least one recombinant nucleic acid substrate molecule encoding the at least one evolved polypeptide with the desired property;
(f) recombining the at least one recombinant nucleic acid substrate molecule of (e) with at least one polynucleotide to produce at least one further evolved nucleic acid substrate molecule;
(g) expressing the at least one further evolved nucleic acid substrate molecule to provide at least one further evolved polypeptide; and
, (h) screening or selecting the at least one further evolved polypeptide for 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
81 Claims
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1. A method of screening or selecting a further evolved polypeptide for a desired property, the method comprising:
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(a) providing a mixture of nucleic acid subsequences of two or more parental nucleic acid substrate molecules encoding homologous polypeptides, wherein each parental nucleic acid substrate molecule differs from at least one other parental nucleic acid substrate molecule in at least one nucleotide;
(b) ligating two or more of the nucleic acid subsequences together in an ordered reassembly to produce a library of recombinant nucleic acid substrate molecules encoding varients of the homologous polypeptides, which library comprises a plurality of nucleic acid molecules, each member of the plurality comprising at least one subsequence from at least two of the two or more parental nucleic acid substrate molecules, wherein the plurality of nucleic acid molecules are homologous to one another and to the parental substrate nucleic acid molecules;
(c) expressing the library of recombinant nucleic acid substrate molecules to provide evolved polypeptides;
(d) screening or selecting the evolved polypeptides to identify at least one evolved polypeptide with the desired property;
(e) recovering at least one recombinant nucleic acid substrate molecule encoding the at least one evolved polypeptide with the desired property;
(f) recombining the at least one recombinant nucleic acid substrate molecule of (e) with at least one polynucleotide to produce at least one further evolved nucleic acid substrate molecule;
(g) expressing the at least one further evolved nucleic acid substrate molecule to provide at least one further evolved polypeptide; and
,(h) screening or selecting the at least one further evolved polypeptide for 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, 80)
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30. A method of identifying an evolved polypeptide with a desired property, the method comprising:
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(a) providing a population of nucleic acid subsequences of two or more parental nucleic acid substrate molecules encoding homologous polypeptides, wherein at least one of the two or more parental nucleic acid substrate molecules is a mutagenized variant;
(b) ligating two or more of the nucleic acid subsequences together in an ordered reassembly to produce a library of recombinant nucleic acid subsequences molecules encoding variants of the homologous polypeptides, which library comprises a plurality of nucleic acid molecules, each member of the plurality comprising at least one subsequence from at least two of the two or more parental nucleic acid substrate molecules, wherein the plurality of nucleic acid molecules are homologous to one another and to the parental substrate nucleic acid molecules;
(c) expressing the library of recombinant nucleic acid substrate molecules to provide evolved polypeptides; and
,(d) screening or selecting the evolved polypeptides to identify at least one evolved polypeptide with the desired property. - View Dependent Claims (31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 81)
(e) recovering at least one recombinant nucleic acid substrate molecule encoding the at least one evolved polypeptide with the desired property; and
,(f) recombining the at least one recombinant nucleic acid substrate molecule of (e) with at least one polynucleotide to produce at least one further evolved nucleic acid substrate molecule.
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53. The method of claim 52, wherein the at least one recombinant nucleic acid substrate molecule recovered in (e) comprises a library of recombinant substrate molecules.
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54. The method of claim 52, wherein the at least one recombinant nucleic acid substrate molecule recovered in (e) is subjected to mutagenesis to provide at least one mutagenized nucleic acid substrate molecule.
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55. The method of claim 54, wherein the mutagenesis comprises one or more of:
- recursive sequence recombination, PCR mutagenesis, oligonucleotide-directed mutagenesis, or site-directed mutagenesis.
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81. The method of claim 44, wherein the restriction endonuclease generates non-palindromic ends at cleavage sites.
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56. A method of screening or selecting a further evolved polypeptide for a desired property, the method comprising:
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(a) providing a population of double-stranded nucleic acid subsequences of two or more polynucleotides encoding variants of a polypeptide, wherein each double-stranded nucleic subsequence comprises one or more single-stranded overhangs that promote reassembly of the subsequences by ligation;
(b) ligating two or more of the double-stranded nucleic acid subsequences together in an ordered reassembly to produce a library of recombined nucleic acid sequences encoding evolved variants of the polypeptide;
(c) expressing the recombined nucleic acid sequences to produce the evolved variants of the polypeptide;
(d) screening or selecting the evolved variants of the polypeptide produced in step (c) to identify at least one evolved variant of the polypeptide comprising the desired property;
(e) recovering at least one recombined nucleic acid sequence encoding the at least one evolved variant of the polypeptide comprising the desired property;
(f) recombining the at least one recombined nucleic acid sequence of (e) with at least one nucleic acid substrate molecule to produce at least one further evolved nucleic acid sequence;
(g) expressing the at least one further evolved nucleic acid sequence to provide at least one further evolved polypeptide; and
,(h) screening or selecting the at least one further evolved polypeptide for the desired property. - View Dependent Claims (57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79)
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Specification