US5427908A
Expired / Lapsed
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Recombinant library screening methods
Assignee: Alere San Diego Inc
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Filing: 1990-05-01
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Granted: 1995-06-27
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Expires: 2010-05-01
Abstract
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.
Inventors
- 👤 William J. Dower
- 👤 Steven E. Cwirla
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 08:00
CPC Classifications
C; C40; C40B; C40B40/00; C40B40/02; C07; C07K; C07K16/00; C07K16/005; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/1037
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
215 words
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.
Claim 2
Independent Claim
215 words
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.
Claim 3
Dependent Claim
18 words
The method according to claim 1, wherein the fusion coat protein comprises an antibody heavy chain variable region.
Claim 4
Dependent Claim
18 words
The method according to claim 1, wherein the fusion coat protein comprises an antibody light chain variable region.
Claim 5
Dependent Claim
27 words
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.
Claim 6
Dependent Claim
15 words
The method according to claim 1, wherein expression of the antibody Fab fragment is inducible.
Claim 7
Dependent Claim
49 words
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.
Claim 8
Dependent Claim
30 words
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.
Claim 9
Dependent Claim
25 words
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.
Claim 10
Dependent Claim
13 words
The method of claim 1, wherein said filamentous bacteriophage expression vector is fd.
Claim 11
Dependent Claim
17 words
The method of claim 9, wherein the fusion coat protein comprises an antibody heavy chain variable region.
Claim 12
Dependent Claim
17 words
The method of claim 9, wherein the fusion coat protein comprises an antibody light chain variable region.
Claim 13
Dependent Claim
46 words
The method of claim 1, wherein from 10 7 to 10 9 host cells are transformed by electroporation with 10 7 to 10 9 filamentous bacteriophage expression vectors, wherein said vectors differ from one another with respect to the antibody Fab fragment encoded by each vector.
Claim 14
Dependent Claim
13 words
The method of claim 12, wherein said filamentous bacteriophage expression vector is fd.
Claim 15
Dependent Claim
17 words
The method of claim 13, wherein the fusion coat protein comprises an antibody heavy chain variable region.
Claim 16
Dependent Claim
17 words
The method of claim 13, wherein the fusion coat protein comprises an antibody light chain variable region.
Claim 17
Dependent Claim
27 words
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.
Claim 18
Dependent Claim
23 words
The method of claim 15, wherein said antibody light chain variable region is located at the amino terminus of said fusion coat protein.
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