Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
First Claim
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1. A method for producing a binding protein with a desired property, the method comprising:
- (i) providing a population of DNA fragments comprising at least one polynucleotide encoding all or part of a binding protein;
(ii) recursively recombining the DNA fragments in vitro or in vivo to form a pool of recombined binding protein polynucleotides, which pool of binding protein polynucleotides comprises a library of shuffled DNA sequences wherein recursively recombining the DNA fragments comprises recombining the DNA fragments to form a pool of recombined binding protein polynucleotides;
selecting the pool for the desired property, thereby providing selected binding protein polynucleotides; and
recombining the selected binding protein polynucleotides and (iii) identifying at least one recombined binding protein polynucleotide encoding a binding protein with a desired property.
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Abstract
A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.
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Citations
26 Claims
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1. A method for producing a binding protein with a desired property, the method comprising:
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(i) providing a population of DNA fragments comprising at least one polynucleotide encoding all or part of a binding protein;
(ii) recursively recombining the DNA fragments in vitro or in vivo to form a pool of recombined binding protein polynucleotides, which pool of binding protein polynucleotides comprises a library of shuffled DNA sequences wherein recursively recombining the DNA fragments comprises recombining the DNA fragments to form a pool of recombined binding protein polynucleotides;
selecting the pool for the desired property, thereby providing selected binding protein polynucleotides; and
recombining the selected binding protein polynucleotides and(iii) identifying at least one recombined binding protein polynucleotide encoding a binding protein with a desired property. - View Dependent Claims (2, 3, 4, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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8. A method for producing an antibody with a desired property, the method comprising:
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(i) providing a population of homologous DNA fragments comprising at least one antibody variable region gene, thereby providing a population of variable region gene fragments;
(ii) recombining the variable region gene fragments to form a pool of recombinant variable region genes, which pool of recombinant variable region genes comprises a library of shuffled DNA sequences; and
(iii) joining at least one recombinant variable region gene produced in step (ii) with at least one additional antibody gene segment to form a recombined antibody polynucleotide encoding an antibody with a desired property. - View Dependent Claims (9, 10, 11)
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Specification