Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-14
2002-06-25
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100
Reexamination Certificate
active
06410239
ABSTRACT:
TABLE OF CONTENTS
1. INTRODUCTION . . . 1
2. BACKGROUND OF THE INVENTION . . . 1
3. SUMMARY OF THE INVENTION . . . 7
4. DESCRIPTION OF THE FIGURES . . . 10
5. DETAILED DESCRIPTION OF THE INVENTION . . . 14
5.1. DETECTING INTERACTING PROTEINS . . . 15
5.2. CHARACTERIZATION OF INTERACTIVE POPULATIONS THAT ARE DIFFERENTIALLY EXPRESSED BY A PARTICULAR TISSUE TYPE, DISEASE STATE OR STAGE OF DEVELOPMENT, AND CREATION OF “PROTEIN INTERACTION MAPS” . . . 39
5.2.1. DETERMINATION OF ALL THE DETECTABLE PROTEIN-PROTEIN INTERACTIONS . . . 40
5.2.2. CLASSIFICATION OF THE ARRAYED POOLS OF INTERACTANTS BY THE QEA™ METHOD AND THE SEQ-QEA™ METHOD . . . 42
5.2.3. ARRAYING AND CODING STRATEGIES FOR AN INTERACTIVE POPULATION . . . 43
5.2.4. MAINTAINING LINKAGE BETWEEN PAIRS OF INTERACTING PROTEINS . . . 44
5.2.5. POOLING STRATEGIES . . . 47
5.2.6. ALTERNATIVE STRATEGIES TO CHARACTERIZE INTERACTIVE POPULATIONS . . . 50
5.2.6.1. SEQUENCE-BASED STRATEGIES TO IDENTIFY PAIRS OF INTERACTING PROTEINS . . . 50
5.2.6.2. CREATION OF INTERACTIVE-GRIDS . . . 51
5.2.7. STATISTICAL CONSIDERATIONS FOR DETECTING ALL POSSIBLE INTERACTIONS AMONG GENES THAT ARE EXPRESSED AT DIFFERENT LEVELS . . . 52
5.2.8. ALTERNATIVE PREFERRED EMBODIMENTS . . . 54
5.2.9. INFORMATION PROCESSING ASPECTS OF DETECTING PROTEIN-PROTEIN INTERACTIONS . . . 69
5.2.9.1. IDENTIFICATION DATABASE AND PROCESSING . . . 74
5.2.9.2. INTERACTION DATABASE . . . 83
5.2.9.3. INTERACTION DATABASE FUNCTIONS . . . 92
5.3. INTEGRATED ISOLATION OF INHIBITORS OF AN INTERACTIVE POPULATION . . . 98
5.4. THE QEA™ METHOD . . . 109
5.4.1. QUANTITATIVE EXPRESSION ANALYSIS METHOD, GENERALLY . . . 109
5.4.2. DETAILS OF A QUANTITATIVE EXPRESSION ANALYSIS METHOD . . . 124
5.4.3. RE EMBODIMENTS OF A QEA™ METHOD . . . 133
5.4.3.1. FIRST ALTERNATIVE RE EMBODIMENT . . . 154
5.4.3.2. SECOND ALTERNATIVE RE EMBODIMENT . . . 156
5.4.4. A SEQ-QEA™ EMBODIMENT OF A QEA™ METHOD . . . 159
5.4.5. QEA™ ANALYSIS AND DESIGN METHODS . . . 167
5.4.5.1. QEA™ EXPERIMENTAL ANALYSIS METHODS . . . 168
5.4.5.2. QEA™ EXPERIMENTAL DESIGN METHODS . . . 182
5.4.5.3. THE QEA™ METHOD AMBIGUITY RESOLUTION . . . 189
5.4.6. APPARATUS FOR PERFORMING THE QEA™ METHODS . . . 192
6. EXAMPLES . . . 198
6.1. DESCRIPTION OF PROTOCOLS . . . 199
6.1.1. MATING PROTOCOL . . . 199
6.1.2. TRANSFORMATION PROTOCOL . . . 204
6.1.3. RNA EXTRACTION . . . 204
6.1.4. DNASE TREATMENT . . . 205
6.1.5. MESSENGER RNA PURIFICATION . . . 205
6.1.6. cDNA SYNTHESIS AND CONSTRUCTION OF FUSION-LIBRARIES . . . 205
6.1.7. TRANSFORMATION OF THE REPORTER STRAINS WITH THE BINDING DOMAIN FUSION cDNA LIBRARY AND ACTIVATION DOMAIN cDNA LIBRARY TO CREATE “M” AND “N” POPULATIONS . . . 207
6.1.8. WHOLE CELL PCR . . . 210
6.1.9. RECOVERY OF COLONIES POSITIVE FOR PROTEIN-PROTEIN INTERACTION . . . 216
6.1.10. PRODUCTION OF PCR POOLS FOR CREATION OF PROTEIN INTERACTION MAPS . . . 217
6.1.11. &bgr;-GALACTOSIDASE ASSAYS . . . 218
6.1.12. PROTOCOLS FOR QEA™ METHODS AND SEQ-QEA™ METHODS . . . 219
6.1.12.1. PREFERRED QEA™ RE METHOD . . . 219
6.1.12.1.1. cDNA PREPARATION . . . 219
6.1.12.1.2. PREFERRED RE/LIGASE AND AMPLIFICATION REACTIONS . . . 220
6.1.12.1.3. PREFERRED AUTOMATED RE/LIGASE REACTIONS . . . 222
6.1.12.1.4. ALTERNATIVE RE/LIGASE AND AMPLIFICATION REACTIONS . . . 225
6.1.12.1.5. OPTIONAL POST-AMPLIFICATION STEPS 228
6.1.12.2. PREFERRED METHODS OF A SEQ-QEA™ EMBODIMENT . . . 229
6.1.12.2.1. QEA™ METHOD PREFERRED FOR USE IN A SEQ-QEA™ METHOD . . . 229
6.1.12.2.2. SEQ-QEA™ METHOD STEPS . . . 232
6.1.12.3. PREFERRED QEA™ METHOD ADAPTERS AND RE PAIRS . . . 236
6.1.12.4. FLUORESCENT LABELS FOR QEA™ METHODS . . . 243
6.1.12.5. PREFERRED REACTANTS FOR SEQ-QEA™ METHODS . . . 245
6.1.13. POST MATING VERIFICATION PROTOCOLS . . . 247
6.1.13.1. PLASMID DROP-OUT PROTOCOL . . . 247
6.1.13.2. YEAST MATRIX MATING PROTOCOL . . . 249
6.2. LIBRARIES . . . 252
6.3. CONSTRUCTION OF YEAST STRAINS . . . 253
6.3.1. CONSTRUCTION OF STRAINS N105 AND N106 . . . 254
6.3.2. CONSTRUCTION OF THE REPORTER STRAIN N106′ . . . 255
6.3.3. CONSTRUCTION OF THE REPORTER STRAIN N105′ . . . . . . . . 255
6.3.4. CONSTRUCTION OF THE REPORTER STRAIN YULH . . . 256
6.3.5. CONSTRUCTION OF THE YEAST STRAIN N203 . . . 257
6.4. CONSTRUCTION OF FUSION GENES . . . 261
6.5. CONSTRUCTION OF cDNA LIBRARIES IN pASSfiI (GDB) . . . 263
6.6. TRANSFORMATION OF THE REPORTER STRAINS WITH THE pASSfiI AND pACT cDNA LIBRARIES TO CREATE “M” AND “N” POPULATIONS . . . 264
6.7. CONSTRUCTION OF YEAST STRAINS WITH INTEGRATED COPIES OF RAF-GAD . . . 264
6.8. CONSTRUCTION OF PEPTIDE EXPRESSION VECTORS (PEVs) . . . 266
6.9. SELECTION OF PROTEIN-PROTEIN INTERACTIONS FROM A NON-INTERACTING BACKGROUND . . . 267
6.10. SELECTION OF SPECIFIC PROTEIN-PROTEIN INTERACTIONS FROM A BACKGROUND OF OTHER INTERACTING PROTEINS . . . 272
6.11.SELECTION OF INTERACTING PROTEINS FROM AN M×N SCREEN . . . 274
6.11.1. MATING ASSAY . . . 274
6.11.2. WHOLE CELL PCR OF THE POSITIVE COLONIES . . . 275
6.11.3. QEA™ METHOD OF THE PCR PRODUCTS . . .275
6.11.4. CREATION OF TWO-DIMENSIONAL POOLS . . . 276
6.11.5. WHOLE CELL PCR OF THE POOLED CELLS . . . 276
6.11.6. QEA™ METHOD OF THE PCR DERIVED FROM POOLED CULTURES . . . 277
6.11.7. THE SEQ-QEA™ METHOD OF THE PCR DERIVED FROM POOLED CULTURES . . . 278
6.12. IDENTIFICATION OF SPECIFIC PAIRS OF INTERACTING PROTEINS FROM A QEA™ METHOD OF THE INTERACTIVE POPULATION AND BY THE USE OF GENE-SPECIFIC PRIMERS . . . 279
6.13. CREATION OF INTERACTIVE GRIDS . . . 279
6.14. ISOLATION OF STAGE-SPECIFIC PAIRS OF INTERACTING PROTEINS . . . 280
6.15. EXPRESSION OF PEPTIDE INHIBITORS IN PEV AND INHIBITION OF PROTEIN-PROTEIN INTERACTIONS . . . 280
6.16. IDENTIFICATION OF CELLS CONTAINING AN INHIBITOR OF PROTEIN-PROTEIN
1. INTRODUCTION
The present method relates to the identification of protein-protein interactions and inhibitors of these interactions that, preferably, are specific to a cell type, tissue type, stage of development, or disease state or stage.
2. BACKGROUND OF THE INVENTION
Proteins and protein-protein interactions play a central role in the various essential biochemical processes. For example, these interactions are evident in the interaction of hormones with their respective receptors, in the intracellular end extracellular signaling events mediated by proteins, in enzyme substrate interactions, in intracellular protein trafficking, in the formation of complex structures like ribosomes, viral coat proteins, and filaments, and in antigen-antibody interactions. These interactions are usually facilitated by the interaction of small regions within the proteins that can fold independently of the rest of the protein. These independent units are called protein domains. Abnormal or disease states can be the direct result of aberrant protein-protein interactions. For example, oncoproteins can cause cancer by interacting with and activating proteins responsible for cell division. Protein-protein interactions are also central to the mechanism of a virus recognizing its receptor on the cell surface as a prelude to infection. Identification of domains that interact with each other not only leads to a broader understanding of protein-protein interactions, but also aids in the design of inhibitors of these interactions.
Protein-protein interactions have been studied by both biochemical and genetic methods. The biochemical methods are laborious and slow, often involving painstaking isolation, purification, sequencing and further biochemical characterization of the proteins being tested for interaction. As an alternative to the biochemical approaches, genetic approaches to detect protein-protein interactions have gained in popularity as these methods allow the rapid detection of the domains involved in protein-protein interactions.
An example of a genetic system to detect protein-protein interactions is the “Two-Hybrid” system to detect protein-protein interactions in the yeast
Saccharomyces cerevisiae
(Fields and Song, 1989, Nature 340:245-246; U.S. Pat. No. 5,283,173 by Fields and Song). This assay utilizes the reconstitution of a transcriptional
Kalbfleisch Theodore Samuel
Knight James Robert
Nandabalan Krishnan
Rothberg Jonathan Marc
Yang Meijia
Campbell Eggerton A.
Chunduru Suryaprabha
CuraGen Corporation
Pennie & Edmonds LLP
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