Patent ID: 6235469
Filing Date: 2001-05-22
Classification: C07K,C12N,C12P,C40B

Abstract:
A method for producing a library of dicistronic DNA molecules, each dicistronic DNA molecule comprising first and second cistrons for expressing first and second polypeptides of a heterodimeric antibody of the surface of a filamentous phage, which method comprises:(a) forming a first ligation admixture by combining in a ligation buffer;(i) a repertoire of first polypeptide genes in the form of linear dsDNA, each having cohesive termini adapted for directional ligation; and(ii) a plurality of DNA expression vectors in linear form, each vector having upstream and downstream first cohesive termini that are (a) adapted for directionally receiving one of said first polypeptide genes in a common reading frame, and (b) operatively linked to respective upstream and downstream translatable DNA sequences, said upstream translatable DNA sequence encoding a procaryotic secretion signal, said downstream translatable DNA sequence encoding a filamentous phage membrane cpVIII anchor domain, and said translatable DNA sequences operatively linked to respective upstream and downstream expression control sequences; and(b) subjecting said admixture to ligation conditions for a time period sufficient to operatively link said first polypeptide genes to said vectors and produce a plurality of circular DNA molecules each having said first cistron for expressing said first polypeptide;(c) treating said plurality of circular DNA molecules produced in step (b) under endonucleolytic conditions sufficient to produce a plurality of DNA expression vectors in linear form, each vector having upstream and downstream second cohesive termini that are (i) adapted for directionally receiving one of a repertoire of second polypeptide genes in a common reading frame, and (ii) operatively linked to respective upstream and downstream translatable DNA sequences, said upstream translatable DNA sequence encoding a procaryotic secretion signal, said downstream DNA sequence having at least one stop codon is said reading frame, and said translatable DNA sequences operatively linked to said expression control sequences;(d) forming a second ligation admixture by combining in a ligation buffer:(i) said plurality DNA expression vectors formed in step (c); and(ii) said repertoire of second polypeptide genes in the form of dsDNA, each having cohesive termini adapted for directional ligation to said plurality of DNA expression vectors formed in step (c); and(e) subjecting said second admixture to ligation conditions for a time period sufficient to operatively link said second polypeptide genes to said vectors and produce a plurality of circular DNA molecules each having said second cistron for expressing said second polypeptide, thereby forming said library.