Peptide library and screening method
AssigneeAffymax Technologies N.V.
InventorsPeter J. Schatz, Willem P. C. Stemmer
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Abstract
A random peptide library constructed by transforming host cells with a collection of recombinant vectors that encode a fusion protein comprised of a DNA binding protein and a random peptide and also contain a binding site for the DNA binding protein can be used to screen for novel ligands. The screening method results in the formation of a complex comprising the fusion protein bound to a receptor through the random peptide ligand and to the recombinant DNA vector through the DNA binding protein.
Claims13
A method for generating and screening a random peptide library, said method comprising the steps of:
- (a) transforming host cells with at least 10.sup.6 different vectors, each of which (i) encodes a fusion protein composed of a random peptide linked to a DNA binding protein consisting essentially of two lac headpieces joined by a linker;
- and (ii) contains a lacO or a derivative lacO binding site, and culturing the resultant transformed host cells under conditions such that said fusion protein is produced and binds to a vector that encodes the fusion protein;
- (b) lysing the cells transformed in step (a) under conditions such that the fusion protein remains bound to the vector that encodes the fusion protein;
- (c) contacting the fusion proteins with a receptor under conditions conducive to specific peptide-receptor binding;
- and
- (d) isolating the vector that encodes a peptide that binds to said receptor.
A mixture of recombinant host cells transformed with a random peptide library composed of at least 10.sup.6 different members, wherein each member is a recombinant DNA vector that (i) encodes a DNA binding protein consisting essentially of two lac headpieces joined by a linker;
- (ii) contains a lacO or derivative lacO binding site;
- and (iii) comprises a coding sequence for a random peptide inserted into the coding sequence of the DNA binding protein such that the resulting vector encodes a fusion protein that is composed of the DNA binding protein and the random peptide;
- and wherein each different member differs from other members with respect to the coding sequence of the random peptide.
A mixture of recombinant host cells transformed with a ligand fragment library composed of at least 10 different members, wherein each member is a recombinant DNA vector that (i) encodes a DNA binding protein consisting essentially of two lac headpieces joined by a linker;
- (ii) contains a lacO or derivative lacO binding site;
- and (iii) comprises a coding sequence for a ligand fragment inserted into the coding sequence of the DNA binding protein, such that the resulting vector encodes a fusion protein that is composed of the DNA binding protein and the ligand fragment;
- and wherein each different member differs from other members with respect to the coding sequence of the ligand fragment.
A recombinant DNA vector comprising:
- (a) a DNA sequence encoding a DNA binding protein consisting essentially of two lac headpieces joined by a linker;
- (b) a promoter positioned so as to drive transcription of said DNA binding protein coding sequence;
- (c) a lacO or derivative lacO binding site;
- and
- (d) a coding sequence for a peptide inserted in said DNA binding protein coding sequence so that said coding sequences can be transcribed to produce a fusion protein composed of said peptide and said DNA binding protein capable of binding to said binding site.
Dependencies are inferred from the claim text.