Methods and compositions for polypeptide engineering
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 producing and isolating a polypeptide having at least one desirable property comprising:
subjecting a starting polynucleotide set to a mutagenesis process so as to produce a set of progeny polynucleotides;
subjecting said progeny polynucleotide set to an end selection-based screening and enrichment process so as to select for a desirable subset of the progeny polynucleotide set, said end-selection process resulting in ligation compatible ends that can be used to facilitate one or more intermolecular ligations by members of the progeny polynucleotide set;
producing a polypeptide set by expression cloning, comprising ligating members of said progeny polynucleotide set into an expression vector using said ligation compatible ends; and
screening said polypeptide set for said at least one desirable property to identify polypeptides comprising said desirable property.
275 . The method of claim 274 , wherein said end selection-based process comprises a selection marker containing a topoisomerase recognition site.
276 . The method of claim 275 , wherein said topoisomerase recognition site comprises a topoisomerase I recognition site.
277 . The method of claim 276 , wherein said topoisomerase I recognition site comprises a vaccinia topoisomerase recognition site.
278 . The method of claim 274 , wherein said mutagenesis and end-selection can be performed iteratively, and in any order, combination and permutation.
279 . The method of claim 274 , wherein said end selection-based process comprises a selection marker containing a restriction enzyme recognition site having at least 6 defined bases.
280 . The method of claim 279 , wherein said restriction enzyme recognition site is selected from the group consisting of BamH I, Bgl II, Psi I, Xba I, Asc I, Not I, Pac I, Pme I, Srf I, Sse838 I, Swa I, BspG I, Eco57 I, and any combination thereof.
281 . The method of claim 274 , wherein said end selection-based process comprises a selection marker containing a restriction enzyme recognition site having at least 8 defined bases.
282 . The method of claim 281 , wherein said restriction enzyme recognition site is selected from Asc I, Not I, Pac I, Pme I, Srf I, Swa I, and any combination thereof.
283 . The method of claim 274 , wherein said end selection-based process comprises a selection marker containing a restriction enzyme recognition site for a Type II or Type IIS restriction enzyme.
284 . The method of claim 274 , wherein the restriction enzyme recognition site comprises a Eco57 I recognition site.