Recombinant library screening methods
AssigneeAffymax Technologies N.V.
InventorsWilliam J. Dower, Steven E. Cwirla
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Abstract
Nucleotide sequences encoding proteins of interest are isolated from DNA libraries using bacteriophage to link the protein to the sequence which encodes it. DNA libraries are prepared from cells encoding the protein of interest and inserted into or adjacent to a coat protein of a bacteriophage vector, or into a sequence encoding a protein which may be linked by means of a ligand to a phage coat protein. By employing affinity purification techniques the phage particles containing sequences encoding the desired protein may be selected and the desired nucleotide sequences obtained therefrom. Thus, for example, novel proteins such as monoclonal antibodies may be produced and conventional hybridoma technology avoided.
Claims18
A method for screening a DNA library for nucleotide sequences which encode a FAB fragment comprising first and second polypeptide chains, one of which chains comprises a heavy chain variable region and the other of which chains comprises a light chain variable region, which FAB fragment binds specifically to a ligand of interest, said method comprising:
- (a) effecting filamentous bacteriophage expression vector transformation of a host cell with:
- (i) a first nucleotide sequence that encodes a fusion coat protein comprising of the first polypeptide chain fused to a coat protein of the bacteriophage;
- and
- (ii) a second nucleotide sequence that encodes the second polypeptide chain fused to a signal peptide that directs periplasmic secretion of said second polypeptide chain;
- (b) cultivating the transformed host cell under conditions suitable for expression and assembly of a bacteriophage particle with a coat comprising said fusion coat protein bound to said second polypeptide chain to form said FAB fragment on the coat of said bacteriophage particle;
- and
- (c) selecting said bacteriophage particle encoding the FAB fragment that binds specifically to the ligand of interest by binding said bacteriophage particle to the ligand specific for said FAB fragment and removing particles that do not bind to said ligand.
A filamentous phage displaying a FAB fragment comprising first and second polypeptide chains, one or which comprises a heavy chain variable region and the other a light chain variable region, which FAB fragment binds specifically to a ligand of interest;
- wherein the first chain is fused to a coat protein on the outersurface of the phage and the second chain is complexed with the first chain.
Dependencies are inferred from the claim text.