Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1995-06-05
2001-01-09
Carlson, Karen Cochrane (Department: 1652)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S385000
Reexamination Certificate
active
06172039
ABSTRACT:
TABLE OF CONTENTS
1. FIELD OF THE INVENTION . . .
2. BACKGROUND OF THE INVENTION . . .
2.1. USE OF HEMOGLOBIN AS A BLOOD SUBSTITUTE
2.1.1. EXPRESSION OF RECOMBINANT HEMOGLOBIN
2.1.2. CHEMICAL MODIFICATION OF HEMOGLOBIN
2.1.3. HEMOGLOBIN VARIANTS
2.2. EXPRESSION OF HETEROLOGOUS DNA IN YEAST
3. SUMMARY OF THE INVENTION
4. BRIEF DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. ISOLATION AND CLONING OF GLOBIN
5.2. GLOBIN VARIANTS
5.3. EXPRESSION OF HEMOGLOBIN
5.3.1. EXPRESSION OF HEMOGLOBIN IN YEAST
5.4. USES FOR EXPRESSED RECOMBINANT HEMOGLOBINS
6. EXAMPLE 1: EXPRESSION OF NATURAL BETA-GLOBIN IN A YEAST EXPRESSION VECTOR CONTAINING GAL10 PROMOTER AND ADH1 TERMINATOR
6.1. MATERIALS
6.2. CLONING OF THE BETA-GLOBIN GENE INTO THE YEAST EXPRESSION VECTOR YEp51
6.3. CLONING OF THE ADH1-TERMINATOR SEQUENCES INTO YEpWB51/NAT
6.4. TRANSFORMATION OF YEAST STRAIN
Sc340 WITH YEp51T/NAT
6.5. QUANTITATION OF RNA FROM SC340 CELLS TRANSFORMED WITH PLASMIDS YEp51, YEp51T/NAT
6.6. WESTERN BLOT ANALYSIS OF EXPRESSED BETA-GLOBIN
7. EXAMPLE 2: CLONING OF ALPHA-GLOBIN INTO A YEAST EXPRESSION VECTOR
7.1. MATERIALS
7.2. ISOLATION OF THE ALPHA-GLOBIN GENE
7.3. PREPARATION OF THE YEAST EXPRESSION VECTOR
7.4. LIGATION AND TRANSFORMATION
7.5. DNA SEQUENCING
8. EXAMPLE 3: EXPRESSION OF NATURAL GAMMA-GLOBIN IN A YEAST EXPRESSION VECTOR CONTAINING GAL10 PROMOTER AND ADH1 TERMINATOR
8.1. MATERIALS
8.2. CLONING OF THE GAMMA-GLOBIN GENE INTO THE YEAST EXPRESSION VECTOR YEp51T/NAT
8.3. TRANSFORMATION AND GROWTH OF YEAST STRAIN Sc340 CELLS WITH PLASMID YEp51T/G
8.4. WESTERN BLOT ANALYSIS OF EXPRESSED GAMMA-GLOBIN
9. EXAMPLE 4: CLONING OF EPSILON-GLOBIN cDNA IN A YEAST EXPRESSION VECTOR AND EXPRESSION OF EPSILON-GLOBIN IN YEAST
9.1. MATERIALS
9.2. SYNTHESIS OF EPSILON-GLOBIN cDN
9.3. CLONING OF EPSILON-GLOBIN cDNA WITH YEAST EXPRESSION VECTOR YEp51NT1
9.4. TRANSFORMATION OF YEAST STRAIN Sc1041 WITH YEp51T/&egr;3
9.5. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN
10. EXAMPLE 5: CLONING OF ZETA-GLOBIN cDNA INTO A YEAST EXPRESSION VECTOR AND EXPRESSION OF ZETA-GLOBIN IN YEAST
10.1. MATERIALS
10.2. CLONING OF ZETA-GLOBIN cDNA IN YEAST EXPRESSION VECTOR pYES2
10.3. TRANSFORMATION OF YEAST STRAIN Sc1041 WITH YES2-&zgr;2
10.4. WESTERN BLOT ANALYSIS OF EXPRESSED-GLOBIN
11. EXAMPLE 6: EXPRESSION OF VARIANT GLOBINS
11.1. MATERIALS AND METHODS
11.1.1. DNA FRAGMENT ISOLATION
11.1.2. DNA LIGATION AND
E. COLI
TRANSFORMATION
11.1.3. PLASMID DNA ANALYSIS
11.1.4. YEAST TRANSFORMATION
11.2. SYNTHESIS OF OLIGONUCLEOTIDES
11.3. IN VITRO MUTAGENESIS
11.3.1. STRAINS
11.3.2. TRANSFECTION OF CJ236
11.3.3. ISOLATION OF URACIL CONTAINING DNA
11.3.4. KINASING OF OLIGONUCLEOTIDES
11.3.5. SYNTHESIS OF THE MUTAGENIC STRAND
11.3.6. TRANSFECTION OF MV1190 CELLS
11.3.7. ANALYSIS OF TRANSFORMANTS BY SEQUENCING
11.4. CONSTRUCTION OF PLASMID YEp51NT1
11.5. CLONING OF VARIANT GLOBINS
11.5.1. CLONING OF PORTO ALEGRE (9 Ala->Cys) &ggr;-GLOBIN GENE
11.5.2. CLONING OF THE MOTOWN (127 Gln->Glu) &ggr;-GLOBIN GENE
11.5.3. CLONING OF THE TITUSVILLE (94 Asp-Asn)&agr;-GLOBIN GENE
11.5.4. CLONING OF THE &bgr;-MISSISSIPPI (44 Ser->Cys) &bgr;-GLOBIN GENE
11.5.5. CLONING OF 104-Ser (104 Cys->Ser) ALPHA-GLOBIN GENE
11.5.6. CLONING OF 104-Ser (104 Cys->Ser) ZETA-GLOBIN GENE
11.5.7. CLONING OF TITUSVILLE (94 Asp-Asn) &zgr;-GLOBIN GENE
11.5.8. CLONING OF ALPHA-GLOBIN GENE CONTAINING ALPHA TITUSVILLE AND ALPHA-104 Ser MUTATIONS: 94 Asp-Asn; 104 Cys->Ser)
11.5.9. CLONING OF THE MOTOWN GAMMA-GLOBIN GENE: (127 Gln->Glu)
11.5.10. CLONING OF THE BOVI2 &bgr;-GLOBIN GENE (Met Leu Thr Ala Glu Glu . . . )
11.5.11. CLONING OF &bgr;-2 Arg GLOBIN GENE: (2 His-Arg)
11.5.12. CLONING OF THE 143 Arg BETA-GLOBIN GENE: (143 His-Arg)
11.5.13. CLONING OF THE 145 Term BETA-GLOBIN GENE: (145 Tyr->TAA)
11.5.14. CLONING OF THE CHICO (66 Lys->Thr) &ggr;-GLOBIN GENE
11.5.15. CLONING OF THE CHICO (66 Lys->Thr) &bgr;-GLOBIN GENE
11.5.16. CLONING OF THE RAINIER (145 Tyr->Cys) &bgr;-GLOBIN GENE
11.5.17. CLONING OF THE TALI (83 GLY->CYS) &bgr;-GLOBIN GENE
11.6. EXPRESSION OF VARIANT GLOBINS
11.6.1. EXPRESSION OF GAMMA-GLOBIN MOTOWN IN A YEAST
11.6.1.1. TRANSFORMATION OF YEAST STRAIN Sc1114 WITH pNT1/&ggr;-Mot2
11.6.1.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN
11.6.2. EXPRESSION OF BETA-GLOBIN Bov2 IN YEAST
11
.
6
.
2
.
1
. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN
11
.
6
.
3
. EXPRESSION OF ZETA-GLOBIN 104 SER IN YEAST
11.6.3.1. TRANSFORMATION OF YEAST STRAIN Sc340 WITH pNT1/Z104S
11.6.3.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN
11.6.4. EXPRESSION OF BETA-GLOBIN TaLi IN YEAST
11.6.5. EXPRESSION OF GAMMA-GLOBIN PORTO ALEGRE IN YEAST
11.6.6. EXPRESSION OF GAMMA-GLOBIN CHICO IN YEAST
11.6.7. EXPRESSION OF BETA-GLOBIN MISSISSIPPI IN YEAST
11.6.8. EXPRESSION OF BETA-GLOBIN RAINIER IN YEAST
11.6.9. EXPRESSION OF BETA-GLOBIN MOTOWN IN YEAST
12. EXAMPLE 7: COEXPRESSION OF ALPHA-GLOBIN AND BETA-GLOBIN BOV2 IN YEAST
12.1. TRANSFORMATION OF YEAST STRAINS Sc389 WITH pUT/2A AND pNT1/&bgr;-Bov2
12.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN
12.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE DIFFERENCE SPECTRUM
13. EXAMPLE 8: COEXPRESSION OF ALPHA-GLOBIN AND BETA-GLOBIN 143 ARG IN YEAST
13.1. TRANSFORMATION OF YEAST STRAIN Sc340 WITH pUT/2A AND pNT1/&bgr;143Arg
13.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN
13.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE DIFFERENCE SPECTRUM
14. EXAMPLE 9: COEXPRESSION OF ALPHA-GLOBIN AND BETA-GLOBIN 145 TERM IN YEAST
14.1. TRANSFORMATION OF YEAST STRAIN Sc340 WITH pUT/2A AND pNT1/&bgr;145T
14.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN
14.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE DIFFERENCE SPECTRUM
14.4. WESTERN BLOT ANALYSIS OF EXPRESSED ALPHA AND BETA GLOBINS
15. EXAMPLE 10: COEXPRESSION OF ALPHA-GLOBIN AND GAMMA-GLOBIN MOTOWN IN YEAST
15.1. TRANSFORMATION OF YEAST STRAIN Sc1114 WITH pUT/2A AND pNT1/&bgr;-Mot2
15.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN
15.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE DIFFERENCE SPECTRUM
16. EXAMPLE 11: COEXPRESSION OF ZETA-GLOBIN 104 SERINE AND BETA-GLOBIN IN YEAST
16.1. TRANSFORMATION OF YEAST STRAIN Sc1114 WITH pNT1/Z104S AND YEp51T/NAT
16.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN
16.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE DIFFERENCE SPECTRUM
17. EXAMPLE 12: COEXPRESSION OF ALPHA-GLOBIN AND BETA-GLOBIN 2 ARG IN YEAST
17.1. TRANSFORMATION OF YEAST STRAIN Sc1090 WITH pUT/2A AND pNT1/&bgr;2Arg
17.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN
17.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE DIFFERENCE SPECTRUM
18. EXAMPLE 13: EXPRESSION OF HEMOGLOBIN PORTLAND I IN YEAST
18.1. TRANSFORMATION OF YEAST STRAIN Sc1012 WITH pYES2-&zgr;2 AND YEp51T/G
18.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN
18.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE DIFFERENCE SPECTRUM
19. EXAMPLE 14: EXPRESSION OF BETA-GLOBIN IN A YEAST EXPRESSION VECTOR CONTAINING A HYBRID PROMOTER AND ADH1 TRANSCRIPTION TERMINATION SEQUENCE
19.1. MATERIALS
19.2. CONSTRUCTION OF PLASMID L19&bgr;t CONTAINING BETA-GLOBIN GENE AND THE ADH1 TERMINATOR
19.3. CONSTRUCTION OF PLASMID pUC19-H&bgr;At
19.4. CLONING OF GAL1-10 UAS INTO pUC19-H&bgr;At
19.5. CLONING OF THE HYBRID PROMOTER-BETA-GLOBIN GENE CASSETTE IN SHUTTLE VECTOR, YEp13
19.6. TRANSFORMATION OF YEAST STRAIN Sc340 CELLS WITH pNML-V-G-1
19.7. WESTERN BLOT ANALYSIS OF EXPRESSED BETA-GLOBIN
20. EXAMPLE 15: EXPRESSION OF HEMOGLOBIN PORTLAND II IN YEAST
20.1. TRANSFORMATION OF YEAST STRAIN Sc1041 WITH pYES2-&zgr;2 AND pNML-V-G-1
20.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN
20.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE DIFFERENCE SPECTRUM
21. EXAMPLE 16: COEXPRESSION OF ALPHA-GLOBIN AND BETA-GLOBIN MISSISSIPPI IN YEAST
21.1. TRANSFORMATION OF YEAST STRAIN Sc389 WITH pUT/2A AND pNT1/&bgr;Miss
21.2. WESTERN BLOT ANALYSIS OF EXPRESSED GLOBIN
21.3. DETECTION OF HEMOGLOBIN IN YEAST BY CARBON MONOXIDE DIFFERENCE SPECTRUM
22. EXAMPLE 16: COEXPRESSION OF ALPHA-GLOBIN TITUSVILLE AND BETA-GLOBIN IN YEAST
22.1. TRANSFORMATION OF YEAST STRAIN Sc1114 WITH pNT1/2ATit and YEp51T/NAT
22.2. WESTERN BL
Bajwa Wajeeh
Bonaventura Joseph
De Angelo Joseph
Motwani Nalini M.
Apex Bioscience, Inc.
Carlson Karen Cochrane
Pennie & Edmonds LLP
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