Recombinant library screening methods

• US 5,427,908 A
• Filed: 05/01/1990
• Issued: 06/27/1995
• Est. Priority Date: 05/01/1990
• Status: Expired due to Term

• Current Assignee: BIOSITE INCORPORATED (Abbott Laboratories Incorporated)
• Original Assignee: Affymax Incorporated
• Inventors: Dower, William J., Cwirla, Steven E.
• Primary Examiner(s): Hutzell, Paula K.
• Assistant Examiner(s): Feisee, Lila
• Application Number:
  US07/517,659
• Time in Patent Office: 1,883 Days
• Field of Search: 435/69.6, 435/6, 435/7.1, 435/5, 435/252.3, 435/69.7, 530/387, 530/387.1, 530/412
• US Class Current: 506/1
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

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.

Claims

1. A method for screening a DNA library for nucleotide sequences which encode an antibody Fab fragment comprising first and second polypeptide chains, one chain comprising a light chain variable region and another chain comprising a heavy chain variable region, wherein said antibody Fab fragment binds specifically to a ligand of interest, said method comprising:

(a) transforming a host cell with a filamentous bacteriophage expression vector selected from the group consisting of fl, fd, and M13 filamentous bacteriophage expression vectors, which vector comprises;

(i) a first nucleotide sequence that encodes a fusion coat protein composed of the first chain fused to a pIII coat protein of the bacteriophage; and

(ii) a second nucleotide sequence that encodes the second chain fused to a signal peptide that directs periplasmic secretion of said second 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 chain to form said antibody Fab fragment on the coat of said particle; and

(c) selecting a bacteriophage particle encoding the antibody Fab fragment that binds specifically to the ligand of interest by binding said particle to the ligand specific for said antibody Fab fragment and removing particles that do not bind to said ligand.

2. The method according to claim 1, wherein the fusion coat protein comprises an antibody heavy chain variable region.

3. The method according to claim 1, wherein the fusion coat protein comprises an antibody light chain variable region.

4. The method according to claim 1, wherein the bacteriophage are isolated from the host cell after the expression and assembly of bacteriophage particles before the selecting step.

5. The method according to claim 1, wherein expression of the antibody Fab fragment is inducible.

6. The method according to claim 1, wherein the transformed host cell is lysed after cultivation and bacteriophage particles are isolated from cellular debris and then selected by binding said particles to a ligand specific for said antibody Fab fragment and removing particles that do not bind to said ligand.

7. The method according to claim 6, wherein the bacteriophage particles which contain the nucleotide sequences encoding the antibody Fab fragment are enriched by repeating the selection step at least once.

8. The method of claim 1 further comprising the step of isolating the nucleotide sequences which encode the antibody Fab fragment from the selected bacteriophage particle.

9. The method of claim 1, wherein said filamentous bacteriophage expression vector is fd.

10. The method of claim 9, wherein the fusion coat protein comprises an antibody heavy chain variable region.

11. The method of claim 9, wherein the fusion coat protein comprises an antibody light chain variable region.

12. The method of claim 1, wherein from 10⁷ to 10⁹ host cells are transformed by electroporation with 10⁷ to 10⁹ filamentous bacteriophage expression vectors, wherein said vectors differ from one another with respect to the antibody Fab fragment encoded by each vector.

13. The method of claim 12, wherein said filamentous bacteriophage expression vector is fd.

14. The method of claim 13, wherein the fusion coat protein comprises an antibody heavy chain variable region.

15. The method of claim 13, wherein the fusion coat protein comprises an antibody light chain variable region.

16. The method of claim 14, wherein said antibody heavy chain variable region is located at the amino terminus of said fusion coat protein on said bacteriophage surface.

17. The method of claim 15, wherein said antibody light chain variable region is located at the amino terminus of said fusion coat protein.