Method for in vitro molecular evolution of protein function
First Claim
1. A method for generating at least one polynucleotide sequence from a parent polynucleotide sequence encoding at least one protein motif, comprising the steps ofa) digesting the parent polynucleotide sequence with an exonuclease to generate a population of fragments;
- b) contacting said fragments with an undigested template polynucleotide sequence under annealing conditions;
c) amplifying the fragments that anneal to the template in step b) to generate at least one polynucleotide sequence encoding at least one protein motif having altered characteristics as compared to the at least one protein motif encoded by said parent polynucleotide.
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Abstract
The present invention relates to a method for in vitro evolution of protein function. In particular, the method relates to the shuffling of nucleotide segments obtained from exonuclease digestion. The present inventors have shown that polynucleotide fragments derived from a parent polynucleotide sequence digested with an exonuclease can be combined to generate a polynucleofide sequence which encodes for a polypeptide having desired characteristics. This method may be usefully applied to the generation of new antibodies or parts thereof having modified characteristics as compared to the parent antibody.
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Citations
18 Claims
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1. A method for generating at least one polynucleotide sequence from a parent polynucleotide sequence encoding at least one protein motif, comprising the steps of
a) digesting the parent polynucleotide sequence with an exonuclease to generate a population of fragments; -
b) contacting said fragments with an undigested template polynucleotide sequence under annealing conditions;
c) amplifying the fragments that anneal to the template in step b) to generate at least one polynucleotide sequence encoding at least one protein motif having altered characteristics as compared to the at least one protein motif encoded by said parent polynucleotide. - View Dependent Claims (2, 3, 8, 11, 12, 13)
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4. A method for generating at least one polynucleotide sequence from a parent polynucleotide sequence encoding at least one protein motif, in which said polynucleotide sequence has altered sequence at its termini as compared to said parent polynucleotide sequence comprising the steps of
(a) digesting said parent polynucleotide sequence with an exonuclease to generate a population of fragments; -
(b) contacting said fragments with an undigested template polynucleotide sequence which is a variant of said parent polynucleotide sequence, under annealing conditions;
(c) amplifying the fragments that anneal to the template in step b) to generate at least one polynucleotide sequence encoding at least one protein motif encoded by said parent polynucleotide. - View Dependent Claims (6, 7, 9, 10, 14, 15, 16)
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5. A method for generating at least one polynucleotide sequence from a parent polynucleotide sequence encoding at least one protein motif, in which said polynucleotide sequence has altered sequence at its center as compared to said parent polynucleotide sequence comprising the steps of
(a) digesting a population of variant polynucleotide sequences with an exonuclease to generate a population of fragments; -
(b) contacting said fragments with said parent polynucleotide sequence under annealing conditions, said parent polynucleotide sequence being undigested;
(c) amplifying the fragments that anneal to the parent in step b) to generate at least one polynucleotide sequence encoding at least one protein motif encoded by said parent polynucleotide. - View Dependent Claims (17, 18)
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Specification