CA2427305A1 - Isolation of binding proteins with high affinity to ligands - Google Patents
Isolation of binding proteins with high affinity to ligands Download PDFInfo
- Publication number
- CA2427305A1 CA2427305A1 CA002427305A CA2427305A CA2427305A1 CA 2427305 A1 CA2427305 A1 CA 2427305A1 CA 002427305 A CA002427305 A CA 002427305A CA 2427305 A CA2427305 A CA 2427305A CA 2427305 A1 CA2427305 A1 CA 2427305A1
- Authority
- CA
- Canada
- Prior art keywords
- bacterium
- further defined
- target substrate
- candidate
- binding protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102000014914 Carrier Proteins Human genes 0.000 title claims abstract 23
- 108091008324 binding proteins Proteins 0.000 title claims abstract 23
- 239000003446 ligand Substances 0.000 title claims abstract 22
- 238000002955 isolation Methods 0.000 title 1
- 238000000034 method Methods 0.000 claims abstract 83
- 241000894006 Bacteria Species 0.000 claims abstract 44
- 210000001322 periplasm Anatomy 0.000 claims abstract 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract 5
- 238000001943 fluorescence-activated cell sorting Methods 0.000 claims abstract 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract 4
- 241000588724 Escherichia coli Species 0.000 claims abstract 3
- 229920001184 polypeptide Polymers 0.000 claims abstract 3
- 239000000758 substrate Substances 0.000 claims 27
- 230000003197 catalytic effect Effects 0.000 claims 14
- 150000007523 nucleic acids Chemical group 0.000 claims 13
- 102000004169 proteins and genes Human genes 0.000 claims 13
- 108090000623 proteins and genes Proteins 0.000 claims 13
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 10
- 238000006243 chemical reaction Methods 0.000 claims 7
- 108020004414 DNA Proteins 0.000 claims 4
- 238000003776 cleavage reaction Methods 0.000 claims 4
- 230000007017 scission Effects 0.000 claims 4
- 102000004190 Enzymes Human genes 0.000 claims 3
- 108090000790 Enzymes Proteins 0.000 claims 3
- 108020004707 nucleic acids Proteins 0.000 claims 3
- 102000039446 nucleic acids Human genes 0.000 claims 3
- 239000000523 sample Substances 0.000 claims 3
- 238000010367 cloning Methods 0.000 claims 2
- 238000002866 fluorescence resonance energy transfer Methods 0.000 claims 2
- 238000007885 magnetic separation Methods 0.000 claims 2
- 230000001131 transforming effect Effects 0.000 claims 2
- 229910019142 PO4 Inorganic materials 0.000 claims 1
- 239000012634 fragment Substances 0.000 claims 1
- 238000002372 labelling Methods 0.000 claims 1
- 239000010452 phosphate Substances 0.000 claims 1
- 238000010791 quenching Methods 0.000 claims 1
- 230000000171 quenching effect Effects 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract 2
- 230000007812 deficiency Effects 0.000 abstract 1
- 239000007850 fluorescent dye Substances 0.000 abstract 1
- 238000001215 fluorescent labelling Methods 0.000 abstract 1
- 102000037865 fusion proteins Human genes 0.000 abstract 1
- 108020001507 fusion proteins Proteins 0.000 abstract 1
- 238000002823 phage display Methods 0.000 abstract 1
- 238000012216 screening Methods 0.000 abstract 1
- 150000003384 small molecules Chemical class 0.000 abstract 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
Abstract
The invention overcomes the deficiencies of the prior art by providing a rapid approach for isolating binding proteins capable of binding small molecules and peptides via "display-less" library screening. In the technique, libraries of candidate binding proteins, such as antibody sequences, are expressed in soluble form in the periplasmic space of gram negative bacteria, such as Escherichia coli, and are mixed with a labeled ligand. In clones expressing recombinant polypeptides with affinity for the ligand, the concentration of the labeled ligand bound to the binding protein is increased and allows the cells to be isolated from the rest of the library. Where fluorescent labeling of the target ligand is used, cells may be isolated by fluorescence activated cell sorting (FACS). The approach is more rapid than prior art methods and avoids problems associated with the surface-expression of ligand fusion proteins employed with phage display.
Claims (74)
1. A method of obtaining a bacterium comprising a nucleic acid sequence encoding a binding protein capable of binding a target ligand comprising the steps of:
(a) providing a Gram negative bacterium comprising a nucleic acid sequence encoding a candidate binding protein, wherein said binding protein is expressed in soluble form in said bacterium;
(b) contacting said bacterium with a labeled ligand capable of diffusing into said bacterium; and (c) selecting said bacterium based on the presence of said labeled ligand within the bacterium, wherein said ligand and said candidate binding protein are bound in said bacterium.
(a) providing a Gram negative bacterium comprising a nucleic acid sequence encoding a candidate binding protein, wherein said binding protein is expressed in soluble form in said bacterium;
(b) contacting said bacterium with a labeled ligand capable of diffusing into said bacterium; and (c) selecting said bacterium based on the presence of said labeled ligand within the bacterium, wherein said ligand and said candidate binding protein are bound in said bacterium.
2. The method of claim 1, further defined as a method of obtaining a nucleic acid sequence encoding a binding protein capable of binding a target ligand, the method further comprising the step of:
(d) cloning said nucleic acid sequence encoding said candidate binding protein.
(d) cloning said nucleic acid sequence encoding said candidate binding protein.
3. The method of claim 1, wherein said binding protein is expressed in soluble form in the periplasm of said bacterium.
4. The method of claim 3, wherein said nucleic acid sequence encoding a candidate binding protein is further defined as operably linked to a leader sequence capable of directing expression of said candidate binding protein in said periplasm.
5. The method of claim 1, wherein said Gram negative bacterium is an E. coli bacterium.
6. The method of claim 1, further defined as comprising providing a population of Gram negative bacteria.
7. The method of claim 6, wherein said population of bacteria is further defined as collectively capable of expressing a plurality of candidate binding proteins.
8. The method of claim 7, wherein said population of bacteria is obtained by a method comprising the steps of:
(a) preparing a plurality DNA inserts which collectively encode a plurality of different potential binding proteins, and (b) transforming a population of Gram negative bacteria with said DNA inserts.
(a) preparing a plurality DNA inserts which collectively encode a plurality of different potential binding proteins, and (b) transforming a population of Gram negative bacteria with said DNA inserts.
9. The method of claim 6, wherein said population of Gram negative bacteria is contacted with said labeled ligand.
10. The method of claim 1, wherein said candidate binding protein is further defined as an antibody or fragment thereof.
11. The method of claim 1, wherein said candidate binding protein is further defined as a binding protein other than an antibody.
12. The method of claim 1, wherein said candidate binding protein is further defined as an enzyme.
13. The method of claim 1, wherein said candidate binding protein is further defined as not capable of diffusing out of said periplasm in intact bacteria.
14. The method of claim 1, wherein said labeled ligand comprises a peptide.
15. The method of claim 1, wherein said labeled ligand comprises a polypeptide.
16. The method of claim 1, wherein said labeled ligand comprises an enzyme.
17. The method of claim 1 where said labeled ligand comprises a nucleic acid.
18. The method of claim 1, wherein said labeled ligand is further defined as comprising a molecular weight of less than about 20,000 Da.
19. The method of claim 1, wherein said labeled ligand is further defined as comprising a molecular weight of less than about 5,000 Da.
20. The method of claim 1, wherein said labeled ligand is further defined as comprising a molecular weight of greater than 600 Da and less than about 30,000 Da.
21. The method of claim 1, wherein said labeled ligand is further defined as fluorescently labeled.
22. The methods of claim 1, wherein said nucleic acid encoding a candidate binding protein if further defined as capable of being amplified following said selection.
23. The method of claim 1, further comprising treating said bacterium to facilitate said diffusing into said periplasm.
24. The method of claim 23, comprising treating the bacterium with hyperosmotic conditions.
25. The method of claim 23, comprising treating the bacterium with physical stress.
26. The method of claim 24, comprising treating the bacterium with a phage.
27. The method of claim 1, wherein said bacterium is grown at a sub-physiological temperature.
28. The method of claim 27, wherein said sub-physiological temperature is about 25°C
29. The method of claim 1, further comprising removing labeled ligand not bound to said candidate binding protein.
30. The method of claim 1, wherein said selecting comprises FACS.
31. The method of claim 1, wherein said selecting comprises magnetic separation.
32. The method of claim 1, wherein said ligand and said candidate binding protein are reversibly bound in said periplasm.
33. A method of obtaining a bacterium comprising a nucleic acid sequence encoding a catalytic protein catalyzing a chemical reaction involving a target substrate, the method comprising the steps of:
(a) providing a Gram negative bacterium comprising a nucleic acid sequence encoding a candidate catalytic protein, wherein said catalytic protein is expressed in soluble form in said bacterium;
(b) contacting said bacterium with a target substrate capable of diffusing into said bacterium, wherein said candidate catalytic protein catalyzes a chemical reaction involving said target substrate and wherein said chemical reaction yields at least a first substrate product; and (c) selecting said bacterium based on the presence of said first substrate product.
(a) providing a Gram negative bacterium comprising a nucleic acid sequence encoding a candidate catalytic protein, wherein said catalytic protein is expressed in soluble form in said bacterium;
(b) contacting said bacterium with a target substrate capable of diffusing into said bacterium, wherein said candidate catalytic protein catalyzes a chemical reaction involving said target substrate and wherein said chemical reaction yields at least a first substrate product; and (c) selecting said bacterium based on the presence of said first substrate product.
34. The method of claim 33, further defined as a method of obtaining a nucleic acid sequence encoding a catalytic protein catalyzing a reaction with a target substrate, the method further comprising the step of:
(d) cloning said nucleic acid sequence encoding said candidate catalytic protein.
(d) cloning said nucleic acid sequence encoding said candidate catalytic protein.
35. The method of claim 33, wherein said candidate catalytic protein is expressed in soluble form in the periplasm of said bacterium.
36. The method of claim 35, wherein said nucleic acid sequence encoding a candidate catalytic protein is further defined as operably linked to a leader sequence capable of directing expression of said candidate catalytic protein in said periplasm.
37. The method of claim 33, wherein said Gram negative bacterium is an E. coli bacterium.
38. The method of claim 33, further defined as comprising providing a population of Gram negative bacteria.
39. The method of claim 38, wherein said population of bacteria is further defined as collectively capable of expressing a plurality of candidate catalytic proteins.
40. The method of claim 39, wherein said population of bacteria is obtained by a method comprising the steps of:
(a) preparing a plurality DNA inserts which collectively encode a plurality of different candidate catalytic proteins, and (b) transforming a population of Gram negative bacteria with said DNA inserts.
(a) preparing a plurality DNA inserts which collectively encode a plurality of different candidate catalytic proteins, and (b) transforming a population of Gram negative bacteria with said DNA inserts.
41. The method of claim 38, wherein said population of Gram negative bacteria is contacted with said target substrate.
42. The method of claim 33, wherein said candidate catalytic protein is further defined as an enzyme.
43. The method of claim 33, wherein said candidate catalytic protein is further defined as not capable of diffusing out of said periplasm.
44. The method of claim 33, wherein said target substrate comprises a molecule containing a scissile amide bond.
45. The method of claim 33, wherein said target substrate comprises a polypeptide.
46. The method of claim 33, wherein said target substrate comprises a molecule containing a scissile carboxylic ester bond.
47. The method of claim 33, wherein said target substrate comprises a nucleic acid.
48. The method of claim 33, wherein said target substrate comprises a molecule containing a scissile phosphate ester bond.
49. The method of claim 33, wherein said target substrate comprises a molecule containing a scissile sulfonate ester bond.
50. The method of claim 33, wherein said target substrate comprises a molecule containing a scissile carbonate ester bond.
51. The method of claim 33, wherein said target substrate comprises a molecule containing a scissile carbamate bond.
52. The method of claim 33, wherein said target substrate comprises a molecule containing a scissile thioester bond.
53. The method of claim 33, wherein said target substrate is further defined as comprising a molecular weight of less than about 20,000 Da.
54. The method of claim 33, wherein said target substrate is further defined as comprising a molecular weight of less than about 5,000 Da.
55. The method of claim 33, wherein said target substrate is further defined as comprising a molecular weight of less than about 3,000 Da.
56. The method of claim 33, wherein said target substrate is further defined as comprising a molecular weight of greater than about 600 Da and less than about 30,000 Da.
57. The method of claim 33, wherein said first substrate product is further defined as capable of being detected based on the presence of a fluorescent signature.
58. The method of claim 57, wherein said fluorescent signature is absent in said target substrate.
59. The method of claim 58, wherein said fluorescent signature is produced by catalytic cleavage of a scissile bond.
60. The method of claim 59, further defined as comprising use of a FRET
system, said FRET system comprising a fluorophore bound by a scissile bond to at least a first molecule capable of quenching the fluorescence of said fluorophore, wherein cleavage of said scissile bond allows said first molecule to diffuse away from the fluorophore and wherein the fluorescence of said fluorophore becomes detectable.
system, said FRET system comprising a fluorophore bound by a scissile bond to at least a first molecule capable of quenching the fluorescence of said fluorophore, wherein cleavage of said scissile bond allows said first molecule to diffuse away from the fluorophore and wherein the fluorescence of said fluorophore becomes detectable.
61. The method of claim 60, wherein the fluorophore comprises a positive charge allowing the fluorophore to remain associated with the bacterium.
62. The method of claim 57, wherein said target substrate is further defined as comprising a latent fluorescent moiety capable of being released by said chemical reaction involving said target substrate.
63. The method of claim 62, wherein the latent fluorescent moiety released by said cleavage possesses an overall positive charge allowing said moiety to remain associated with the bacterium following said cleavage.
64. The method of claim 57, further defined as comprising labeling said target substrate with a fluorescent pH probe capable of being detected upon a change in pH
associated with said chemical reaction involving said target substrate.
associated with said chemical reaction involving said target substrate.
65. The method of claim 64, wherein said fluorescent pH probe possesses an overall positive charge allowing said fluorescent pH probe to remain associated with the bacterium following said chemical reaction involving said target substrate.
66. The method of claim 33, wherein said bacterium is further defined as viable following said selecting.
67. The method of claim 33, further comprising treating said bacterium to facilitate said diffusing into said periplasm.
68. The method of claim 67, comprising treating the bacterium with hyperosmotic conditions.
69. The method of claim 67, comprising treating the bacterium with physical stress.
70. The method of claim 67, comprising treating the bacterium with a phage.
71. The method of claim 33, wherein said bacterium is grown at a sub-physiological temperature.
72. The method of claim 71, wherein said sub-physiological temperature is about 25°C.
73. The method of claim 33, wherein said selecting comprises FACS.
74. The method of claim 33, wherein said selecting comprises magnetic separation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA2645194A CA2645194C (en) | 2000-10-27 | 2001-10-26 | Isolation of binding proteins with high affinity to ligands |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US09/699,023 US7083945B1 (en) | 2000-10-27 | 2000-10-27 | Isolation of binding proteins with high affinity to ligands |
| US09/699,023 | 2000-10-27 | ||
| PCT/US2001/046795 WO2002034886A2 (en) | 2000-10-27 | 2001-10-26 | Isolation of binding proteins with high affinity to ligands |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2645194A Division CA2645194C (en) | 2000-10-27 | 2001-10-26 | Isolation of binding proteins with high affinity to ligands |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2427305A1 true CA2427305A1 (en) | 2002-05-02 |
| CA2427305C CA2427305C (en) | 2011-10-04 |
Family
ID=24807610
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2645194A Expired - Fee Related CA2645194C (en) | 2000-10-27 | 2001-10-26 | Isolation of binding proteins with high affinity to ligands |
| CA2427305A Expired - Fee Related CA2427305C (en) | 2000-10-27 | 2001-10-26 | Isolation of binding proteins with high affinity to ligands |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2645194A Expired - Fee Related CA2645194C (en) | 2000-10-27 | 2001-10-26 | Isolation of binding proteins with high affinity to ligands |
Country Status (9)
| Country | Link |
|---|---|
| US (2) | US7083945B1 (en) |
| EP (1) | EP1356023B1 (en) |
| JP (2) | JP4159872B2 (en) |
| AT (1) | ATE489453T1 (en) |
| AU (2) | AU2002230635B2 (en) |
| CA (2) | CA2645194C (en) |
| DE (1) | DE60143545D1 (en) |
| DK (1) | DK1356023T3 (en) |
| WO (1) | WO2002034886A2 (en) |
Families Citing this family (25)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7094571B2 (en) * | 2000-10-27 | 2006-08-22 | The Board Of Regents Of The University Of Texas System | Combinatorial protein library screening by periplasmic expression |
| US7083945B1 (en) * | 2000-10-27 | 2006-08-01 | The Board Of Regents Of The University Of Texas System | Isolation of binding proteins with high affinity to ligands |
| EP1495055B1 (en) * | 2002-04-18 | 2013-08-14 | Genencor International, Inc. | Production of functional antibodies in filamentous fungi |
| US7601351B1 (en) | 2002-06-26 | 2009-10-13 | Human Genome Sciences, Inc. | Antibodies against protective antigen |
| US7611866B2 (en) * | 2002-07-15 | 2009-11-03 | Board Of Regents, The University Of Texas System | Selection of bacterial inner-membrane anchor polypeptides |
| US6916474B2 (en) * | 2002-07-15 | 2005-07-12 | Board Of Regents, The University Of Texas System | Antibodies with increased affinities for anthrax antigens |
| US20040067532A1 (en) | 2002-08-12 | 2004-04-08 | Genetastix Corporation | High throughput generation and affinity maturation of humanized antibody |
| US20040248109A1 (en) * | 2003-06-09 | 2004-12-09 | Lawrence Greenfield | Methods for selecting protein binding moieties |
| WO2005103074A2 (en) * | 2004-03-18 | 2005-11-03 | Board Of Regents, The University Of Texas System | Combinatorial protein library screening by periplasmic expression |
| US20060040327A1 (en) * | 2004-08-18 | 2006-02-23 | Terry Amiss | Methods of screening proteins |
| ATE555128T1 (en) * | 2006-11-30 | 2012-05-15 | Res Dev Foundation | IMPROVED IMMUNOLOBULIN LIBRARIES |
| WO2009036379A2 (en) | 2007-09-14 | 2009-03-19 | Adimab, Inc. | Rationally designed, synthetic antibody libraries and uses therefor |
| US8877688B2 (en) | 2007-09-14 | 2014-11-04 | Adimab, Llc | Rationally designed, synthetic antibody libraries and uses therefor |
| WO2010104115A1 (en) * | 2009-03-10 | 2010-09-16 | 独立行政法人産業技術総合研究所 | Method for preparing polypeptide specifically recognizing membrane protein |
| EP2516702B1 (en) * | 2009-12-23 | 2018-03-21 | Affinity Biosciences Pty Ltd | Protein display |
| DK3741883T3 (en) | 2010-07-16 | 2023-02-20 | Adimab Llc | ANTIBODIES LIBRARIES |
| JP6153921B2 (en) * | 2011-03-24 | 2017-06-28 | オプコ ファーマシューティカルズ、エルエルシー | Biomarker discovery in liquid biological samples using bead or particle-based libraries, and diagnostic kits and treatments |
| DK2726652T3 (en) * | 2011-06-29 | 2017-01-23 | Affinity Biosciences Pty Ltd | PROCEDURE FOR PROTEIN INSTRUCTIONS |
| US20140243234A1 (en) * | 2011-08-03 | 2014-08-28 | Texas Biomedical Research Institute | Nucleic acid compositions, methods and kits for rapid pairing of affinity agents |
| SG11201408330XA (en) | 2012-05-24 | 2015-01-29 | Mountgate Group Ltd | Compositions and methods related to prevention and treatment of rabies infection |
| JP2021502125A (en) | 2017-11-09 | 2021-01-28 | ピンテオン セラピューティクス インコーポレイテッド | Methods and Compositions for the Preparation and Use of Humanized Conformation-Specific Phosphorylated Tau Antibodies |
| AU2018383600A1 (en) | 2017-12-11 | 2020-06-18 | Abalone Bio, Inc. | Yeast display of proteins in the periplasmic space |
| JP2023509565A (en) * | 2019-11-27 | 2023-03-09 | イノバシオン・イ・デサロージョ・デ・エネルヒーア・アルファ・スステンタブレ・ソシエダ・アノニマ・デ・カピタル・バリアブレ | phage-resistant microorganisms |
| KR20230146522A (en) | 2021-01-13 | 2023-10-19 | 메모리얼 슬로안 케터링 캔서 센터 | Anti-DLL3 antibody-drug conjugate |
| JP2024503657A (en) | 2021-01-13 | 2024-01-26 | メモリアル スローン－ケタリング キャンサー センター | Antibody-pyrrolobenzodiazepine derivative conjugate |
Family Cites Families (35)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3826364A (en) | 1972-05-22 | 1974-07-30 | Univ Leland Stanford Junior | Particle sorting method and apparatus |
| EP0177343B1 (en) | 1984-10-05 | 1992-07-22 | Genentech, Inc. | Dna, cell cultures and methods for the secretion of heterologous proteins and periplasmic protein recovery |
| US4946773A (en) | 1985-12-23 | 1990-08-07 | President And Fellows Of Harvard College | Detection of base pair mismatches using RNAase A |
| US5223409A (en) | 1988-09-02 | 1993-06-29 | Protein Engineering Corp. | Directed evolution of novel binding proteins |
| US5160974A (en) | 1990-06-25 | 1992-11-03 | Flow Science, Inc. | Closed sample cell for use in flow cytometry |
| US5780279A (en) | 1990-12-03 | 1998-07-14 | Genentech, Inc. | Method of selection of proteolytic cleavage sites by directed evolution and phagemid display |
| DE69230142T2 (en) * | 1991-05-15 | 2000-03-09 | Cambridge Antibody Tech | METHOD FOR PRODUCING SPECIFIC BINDING PAIRS |
| JP3951062B2 (en) | 1991-09-19 | 2007-08-01 | ジェネンテック・インコーポレーテッド | Expression of antibody fragments with cysteine present at least as a free thiol in E. coli for the production of bifunctional F (ab ') 2 antibodies |
| US5866344A (en) * | 1991-11-15 | 1999-02-02 | Board Of Regents, The University Of Texas System | Antibody selection methods using cell surface expressed libraries |
| US5348867A (en) * | 1991-11-15 | 1994-09-20 | George Georgiou | Expression of proteins on bacterial surface |
| DE69334095T2 (en) * | 1992-07-17 | 2007-04-12 | Dana-Farber Cancer Institute, Boston | Method for intracellular binding of targeted molecules |
| US5521066A (en) | 1993-09-13 | 1996-05-28 | Bristol-Myers Squibb Company | Periplasmic membrane-bound system for detecting protein-protein interactions |
| US5922545A (en) | 1993-10-29 | 1999-07-13 | Affymax Technologies N.V. | In vitro peptide and antibody display libraries |
| DE69433901T2 (en) * | 1993-11-18 | 2005-07-28 | Siga Technologies, Inc. | COMPOUNDS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT AND PROPHYLAXIS OF BACTERIAL INFECTIONS |
| GB9408668D0 (en) | 1994-04-30 | 1994-06-22 | Orthosonics Ltd | Untrasonic therapeutic system |
| NZ286241A (en) | 1995-03-29 | 1997-11-24 | Mitsui Chemicals Inc | Method of producing a peptide as a fusion protein intracellularly which is cleaved on passing the cell membrane |
| US5702892A (en) | 1995-05-09 | 1997-12-30 | The United States Of America As Represented By The Department Of Health And Human Services | Phage-display of immunoglobulin heavy chain libraries |
| US5955604A (en) * | 1996-10-15 | 1999-09-21 | The Regents Of The University Of California | Substrates for β-lactamase and uses thereof |
| US6326204B1 (en) * | 1997-01-17 | 2001-12-04 | Maxygen, Inc. | Evolution of whole cells and organisms by recursive sequence recombination |
| IL130635A0 (en) * | 1997-01-17 | 2000-06-01 | Maxygen Inc | Evolution of whole cells and organisms by recursive sequence recombination |
| WO1998049286A2 (en) * | 1997-05-01 | 1998-11-05 | Board Of Regents, The University Of Texas System | Directed evolution of enzymes and antibodies |
| US6342611B1 (en) * | 1997-10-10 | 2002-01-29 | Cytovia, Inc. | Fluorogenic or fluorescent reporter molecules and their applications for whole-cell fluorescence screening assays for capsases and other enzymes and the use thereof |
| US6022952A (en) * | 1998-04-01 | 2000-02-08 | University Of Alberta | Compositions and methods for protein secretion |
| US6902918B1 (en) | 1998-05-21 | 2005-06-07 | California Institute Of Technology | Oxygenase enzymes and screening method |
| DE69920473T2 (en) * | 1998-07-21 | 2006-02-23 | Cytovia, Inc., San Diego | NEW FLUORESCENCE DYES AND THEIR APPLICATION IN FLUORESCENCE DETECTION METHODS OF FULL CELLS FOR CASAS, PEPTIDASES, PROTEASES AND OTHER ENZYMES AND THEIR USE |
| US6180343B1 (en) * | 1998-10-08 | 2001-01-30 | Rigel Pharmaceuticals, Inc. | Green fluorescent protein fusions with random peptides |
| US6391575B1 (en) * | 1999-03-05 | 2002-05-21 | Idun Pharmaceuticals, Inc. | Methods for detecting membrane derived caspase activity and modulators thereof |
| US7033781B1 (en) * | 1999-09-29 | 2006-04-25 | Diversa Corporation | Whole cell engineering by mutagenizing a substantial portion of a starting genome, combining mutations, and optionally repeating |
| AU2000280285A1 (en) | 2000-09-13 | 2002-03-26 | California Institute Of Technology | Oxygenase enzymes and screening method |
| US7083945B1 (en) | 2000-10-27 | 2006-08-01 | The Board Of Regents Of The University Of Texas System | Isolation of binding proteins with high affinity to ligands |
| US7094571B2 (en) * | 2000-10-27 | 2006-08-22 | The Board Of Regents Of The University Of Texas System | Combinatorial protein library screening by periplasmic expression |
| US6916474B2 (en) * | 2002-07-15 | 2005-07-12 | Board Of Regents, The University Of Texas System | Antibodies with increased affinities for anthrax antigens |
| US7611866B2 (en) * | 2002-07-15 | 2009-11-03 | Board Of Regents, The University Of Texas System | Selection of bacterial inner-membrane anchor polypeptides |
| WO2005103074A2 (en) * | 2004-03-18 | 2005-11-03 | Board Of Regents, The University Of Texas System | Combinatorial protein library screening by periplasmic expression |
| WO2005095988A2 (en) | 2004-03-18 | 2005-10-13 | Board Of Regents, The University Of Texas System | Selection of bacterial inner-membrane anchor polypeptides |
-
2000
- 2000-10-27 US US09/699,023 patent/US7083945B1/en not_active Expired - Lifetime
-
2001
- 2001-10-26 CA CA2645194A patent/CA2645194C/en not_active Expired - Fee Related
- 2001-10-26 DE DE60143545T patent/DE60143545D1/en not_active Expired - Lifetime
- 2001-10-26 AT AT01988762T patent/ATE489453T1/en not_active IP Right Cessation
- 2001-10-26 DK DK01988762.9T patent/DK1356023T3/en active
- 2001-10-26 AU AU2002230635A patent/AU2002230635B2/en not_active Ceased
- 2001-10-26 CA CA2427305A patent/CA2427305C/en not_active Expired - Fee Related
- 2001-10-26 EP EP01988762A patent/EP1356023B1/en not_active Expired - Lifetime
- 2001-10-26 AU AU3063502A patent/AU3063502A/en active Pending
- 2001-10-26 JP JP2002537857A patent/JP4159872B2/en not_active Expired - Fee Related
- 2001-10-26 WO PCT/US2001/046795 patent/WO2002034886A2/en active Application Filing
-
2006
- 2006-08-01 US US11/461,757 patent/US7871796B2/en not_active Expired - Fee Related
-
2008
- 2008-01-16 JP JP2008007326A patent/JP2008099710A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| US7871796B2 (en) | 2011-01-18 |
| JP2004531206A (en) | 2004-10-14 |
| EP1356023B1 (en) | 2010-11-24 |
| AU2002230635B2 (en) | 2007-02-08 |
| CA2645194C (en) | 2012-02-07 |
| US7083945B1 (en) | 2006-08-01 |
| DK1356023T3 (en) | 2011-03-14 |
| WO2002034886A3 (en) | 2003-07-31 |
| WO2002034886A9 (en) | 2003-02-13 |
| ATE489453T1 (en) | 2010-12-15 |
| CA2645194A1 (en) | 2002-05-02 |
| CA2427305C (en) | 2011-10-04 |
| EP1356023A2 (en) | 2003-10-29 |
| WO2002034886A2 (en) | 2002-05-02 |
| US20070065913A1 (en) | 2007-03-22 |
| AU3063502A (en) | 2002-05-06 |
| JP4159872B2 (en) | 2008-10-01 |
| DE60143545D1 (en) | 2011-01-05 |
| JP2008099710A (en) | 2008-05-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2427305A1 (en) | Isolation of binding proteins with high affinity to ligands | |
| JP7097627B2 (en) | Large molecule analysis using nucleic acid encoding | |
| Flynn et al. | Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals | |
| JP2021501577A (en) | Kit for analysis using nucleic acid encoding and / or labeling | |
| US20040076966A1 (en) | Method and system for the co-isolation of cognate DNA, RNA and protein sequences and method for screening co-isolates for defined activities | |
| JP2003505041A (en) | In vitro selection using solid support carriers and arbitrary identification of polypeptides | |
| CZ20013399A3 (en) | Protein isolation method and protein analysis, particularly mass spectrometry analysis | |
| WO2014189768A1 (en) | Devices and methods for display of encoded peptides, polypeptides, and proteins on dna | |
| Nord et al. | Microbead display of proteins by cell-free expression of anchored DNA | |
| CN111304753B (en) | Method and kit for screening DNA encoding molecular library | |
| HUP0102457A2 (en) | Novel methods for the identification of ligand and target biomolecules | |
| US20220073904A1 (en) | Devices and methods for display of encoded peptides, polypeptides, and proteins on dna | |
| US7527954B2 (en) | Method for in vitro evolution of polypeptides | |
| JP4303112B2 (en) | Methods for the generation and identification of soluble protein domains | |
| US20030143576A1 (en) | Method and device for integrated protein expression, purification and detection | |
| US8586315B2 (en) | Fluorescent protein particles | |
| JP4122694B2 (en) | Protein-DNA linking molecule and use thereof | |
| US20040265835A1 (en) | Method of sorting vesicle-entrapped, coupled nucleic acid-protein displays | |
| Simon et al. | Application of a Dual Internally Quenched Fluorogenic Substrate in Screening for D-Arginine Specific Proteases | |
| Machova et al. | Expressed protein ligation for protein semisynthesis and engineering | |
| WO2023150742A2 (en) | Methods for generating nucleic acid encoded protein libraries and uses thereof | |
| US20060099713A1 (en) | Targeted-assisted iterative screening (tais):a novel screening format for large molecular repertoires | |
| KR20240042497A (en) | Polypeptide production method, tag, expression vector, polypeptide evaluation method, nucleic acid display library production method, and screening method | |
| US20010024789A1 (en) | Methods for generating catalytic proteins | |
| JPH06510901A (en) | Universal site-specific nuclease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |
Effective date: 20131028 |