Methods and compositions for polypeptide engineering

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.

1 - 273 . (canceled)  
     
     
         274 . A method for generating a mutagenized polynucleotide comprising: 
 (a) annealing a poly-binding nucleic acid strand to two or more mono-binding nucleic acid strands to generate an annealed heteromeric complex of nucleic acid strands, wherein said annealed complex comprises about 10, 100, 1,000, 10,000, 100,000 or 1,000,000 bases;    (b) subjecting unhybridized single-stranded ends of the annealed mono-binding nucleic acid strands in the heteromeric complex to an exonuclease treatment that degrades said unhybridized ends; and    (c) subjecting the annealed hertomeric complex to polymerase-based extension.    
     
     
         275 . A method for generating a mutagenized polynucleotide comprising: 
 (a) annealing a poly-binding nucleic acid strand to two or more mono-binding nucleic acid strands to generate an annealed heteromeric complex of nucleic acid strands, wherein the mono-binding strands and/or the poly-binding strands are generated from a template progenitor molecule by synthesis, fragmentation, isolation or denaturation;    (b) subjecting unhybridized single-stranded ends of the annealed mono-binding nucleic acid strands in the heteromeric complex to an exonuclease treatment that degrades said unhybridized ends; and    (c) subjecting the annealed heteromeric complex to polymerase-based extension.    
     
     
         276 . A method for generating a mutagenized polynucleotide comprising: 
 (a) annealing a poly-binding nucleic acid strand to two or more mono-binding nucleic acid strands to generate an annealed heteromeric complex of nucleic acid strands, wherein the mono-binding strands and/or the poly-binding strands are derived from a library of clones generated from nucleic acid from a mixed population of organisms;    (b) subjecting unhybridized single-stranded ends of the annealed mono-binding nucleic acid strands in the heteromeric complex to an exonuclease treatment that degrades said unhybridized ends; and    (c) subjecting the annealed heteromeric complex to polymerase-based extension.    
     
     
         277 . The method of  claim 274 , further comprising ligating the annealed and extented heteromeric complex.  
     
     
         278 . The method of  claim 275 , further comprising ligating the annealed and extended heteromeric complex.  
     
     
         279 . The method of  claim 276 , further comprising ligating the annealed and extended heteromeric complex.  
     
     
         280 . The method of  claim 278 , wherein the mono-binding strands and/or the poly-binding strands are generated from a template progenitor molecule by synthesis, fragmentation, isolation or denaturation.  
     
     
         281 . A method for generating a mutant nucleic acid, the method comprising: 
 (a) providing a single-stranded polynucleotide;    (b) providing at least two single-stranded nucleic acid fragments of the single-stranded polynucleotide, each of which differs from the single-stranded polynucleotide in at least one nucleotide and is capable of hybridizing to the single-stranded polynucleotide;    (c) annealing the at least two single-stranded nucleic acid fragments to the single-stranded polynucleotide to generate an annealed nucleic acid product;    (d) contacting the annealed nucleic acid product with an enzyme that digests an unhybridized end(s) of at least one single-stranded nucleic acid fragment; and    (e) subjecting the annealed nucleic acid product to polymerase-based extension.    
     
     
         282 . The method of  claim 281 , wherein the enzyme that digests an unhybridized end(s) of at least one single-stranded nucleic acid fragment is Taq, Klenow, or Vent DNA polymerase.  
     
     
         283 . The method of  claim 281 , further comprising subjecting the annealed nucleic acid product to ligase, thereby ligating single-stranded nucleic acid fragments.  
     
     
         284 . The method of  claim 281 , further comprising subjecting the annealed nucleic acid product to ligase and polymerase-based extension using a DNA polymerase.  
     
     
         285 . The method of  claim 281 , wherein the annealed nucleic acid product comprises about 100 kilobases.  
     
     
         286 . The method of  claim 281 , wherein the at least two single-stranded nucleic acid fragments are produced by fragmentation of at least one nucleic acid that differs from the single-stranded polynucleotide in at least one nucleotide.  
     
     
         287 . The method of  claim 281 , wherein the at least two single-stranded nucleic acid fragments are produced by nucleic acid synthesis.  
     
     
         288 . A method for generating a population of mutant nucleic acids, the method comprising: 
 (a) providing at least one single-stranded polynucleotide;    (b) providing a population of at least two single-stranded nucleic acid fragments of the single-stranded polynucleotide, each of which differs from the at least one single-stranded polynucleotide in at least one nucleotide and is capable of hybridizing to the single-stranded polynucleotide;    (c) annealing the at least two single-stranded nucleic acid fragments to the at least one single-stranded polynucleotide to generate a population of annealed nucleic acid products;    (d) contacting the population of annealed nucleic acid products with an enzyme that digests at least one unhybridized end of at least one single-stranded nucleic acid fragment;    (e) subjecting the population of annealed nucleic acid products to polymerase-based extension; and    (f) subjecting the products of (e) to ligase, thereby generating a population of mutant nucleic acids.    
     
     
         289 . The method of  claim 288 , further comprising denaturing and fragmenting the population of mutant nucleic acids, and repeating steps (a)-(f) using resultant single-stranded mutant nucleic acid fragments as the at least two single-stranded nucleic acid fragments to generate a further population of mutant nucleic acids.  
     
     
         290 . The method of  claim 288 , further comprising selecting or screening the population of mutant nucleic acids or encoded products thereof for a desired property.  
     
     
         291 . The method of  claim 289 , further comprising selecting or screening the further population of mutant nucleic acids or encoded products thereof for a desired property.  
     
     
         292 . The method of  claim 288 , wherein the enzyme is Taq, Klenow, or Vent DNA polymerase.  
     
     
         293 . The method of  claim 288 , further comprising subjecting the population of annealed nucleic acid products to ligase, thereby ligating single-stranded nucleic acid fragments.  
     
     
         294 . The method of  claim 288 , further comprising subjecting the population of annealed nucleic acid products to ligase and polymerase-based extension using a DNA polymerase.  
     
     
         295 . The method of  claim 281 , wherein each of the at least two single-stranded nucleic acid fragments differs from the single-stranded polynucleotide in at least one or two nucleotide positions and shares a region of sequence homology of about 10 to 100 base pairs with the single-stranded polynucleotide.  
     
     
         296 . The method of  claim 281 , wherein each of the at least two single-stranded nucleic acid fragments differs from the single-stranded polynucleotide in at least one or two nucleotide positions, but shares 80 or 90% sequence identity with the single-stranded polynucleotide.  
     
     
         297 . The method of  claim 288 , wherein each of the at least two single-stranded nucleic acid fragments differ from the at least one single-stranded polynucleotide in at least one or two nucleotide positions and shares a region of sequence homology of about 10 to 100 base pairs with the single-stranded polynucleotide.  
     
     
         298 . The method of  claim 288 , wherein each of the at least two single-stranded nucleic acid fragments differs from the at least one single-stranded polynucleotide in at least one or two nucleotide positions, but shares 80 or 90% sequence identity with the single-stranded polynucleotide.