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1. WO1983004030 - FABRICATION ET EXPRESSION DE GENES POUR L'UROGASTRONE ET LEURS ANALOGUES POLYPEPTIDES

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

WHAT IS CLAIMED IS:

1. A manufactured gene capable of directing the synthesis in a selected host microorganism of urogastrone polypeptide.

2. A manufactured gene according to claim

1 wherein the base sequence includes one or more codons, selected from among alternative codons specifying the same amino acid, on the basis of preferential expression characteristics of the codon in a projected host microorganism.

3. A manufactured gene according to claim 1 wherein the base sequence includes one or more codons, selected from among alternative codons specifying the same amino acid, on the basis of preferential expression characteristics of the codon in E_. coli.

4. A manufactured gene according to claim

1 wherein the base sequence comprises one of the following:

5'-AAC TCT GAT TCC GAA TGC CCG CTG Tecs' -TTG AGA CTA AGG CTT ACG GGC GAC AGG-

CAC GAC GGT TAT TGT CTG CAC GAC GGT GTT TGC ATG-GTG CTG CCA ATA ACA GAC GTG CTG CCA CAA ACG TAC-

TAT ATC GAA GCT CTG GAC AAA TAC GCT TGC AAC TGC-ATA TAG CTT CGA GAC CTG TTT ATG CGA ACG TTG ACG-

GTA GTT GGT TAC ATC GGT GAA CGT TGC CAG TAC CGT-CAT CAA CCA ATG TAG CCA CTT GCA ACG GTC ATG GCA-

GAT CTG AAA TGG TGG GAA CTG CGT-3 '
CTA GAC TTT ACC ACC CTT GAC GCA-5 '

and


5* -AAC TCT GAT TCC GAA TGC CCG CTG TCT CAT GAC GGT 3' -TTG AGA CTA AGG CTT ACG GGC GAC AGA GTA CTG CCA

TAC TGC CTG CAT GAT GGC GTA TGC ATG TAC ATC GAA GCT ATG ACG GAC GTA CTA CCG CAT ACG TAC ATG TAG CTT CGA

CTG GAC AAA TAC GCA TGC AAC TGT GTT GTA GGT TAC ATC GAC CTG TTT ATG CGT ACG TTG ACA CAA CAT CCA ATG TAG

GGC GAA CGT TGC CAG TAT CGC GAC CTG AAA TGG TGG GAA CCG CTT GCA ACG GTC ATA GCG CTG GAC TTT ACC ACC CTT

CTG CGT-3 '
GAC GCA-5'

5. A manufactured gene according to claim

1 wherein base codons specifying urogastrone include initial and/or terminal codons respectively specifying additional initial and/or terminal amino acids in the polypeptide synthesized.

6. A manufactured gene according to claim 1 wherein the base codons specifying urogastrone are preceded and/or followed by a sequence of bases comprising a portion of a base sequence which provides a recognition site for restriction endonuclease enzyme cleavage.

7. A manufactured gene capable of directing the synthesis in a selected host microorganism of a urogastrone polypeptide analog which differs from therefrom in terms of the identity and/or location of one or more amino acids.

8. A manufactured gene according to claim 7 wherein the base sequence includes one or more codons, selected from among alternative codons specifying the same amino acid, on the basis of preferential expression characteristics of the codon in a projected host microorganism.

9. A manufactured gene according to claim 8 wherein the base sequence includes one or more codons, selected from among alternative codons specifying the same amino acid, on the basis of preferential expression characteristics of the codon in E. coli.

10. A manufactured gene according to claim

7 wherein the base sequence comprises one of the following:

5' -ATG AAC TCT GAT TCC GAA TGC CCG CTG Tecs'-TAC TTG AGA CTA AGG CTT ACG GGC GAC AGG-

CAC GAC GGT TAT TGT CTG CAC GAC GGT GTT TGC ATG-GTG CTG CCA ATA ACA GAC GTG CTG CCA CAA ACG TAC-

TAT ATC GAA GCT CTG GAC AAA TAC GCT TGC AAC TGC-ATA TAG CTT CGA GAC CTG TTT ATG CGA ACG TTG ACG-

GTA GTT GGT TAC ATC GGT GAA CGT TGC CAG TAC CGT-CAT CAA CCA ATG TAG CCA CTT GCA ACG GTC ATG GCA- GAT CTG AAA TGG TGG GAA CTG CGT-3 '
CTA GAC TTT ACC ACC CTT GAC GCA-5 '

and

5' -ATG AAC TCT GAT TCC GAA TGC CCG CTG TCT CAT GAC GGT 3' -TAC TTG AGA CTA AGG CTT ACG GGC GAC AGA GTA CTG CCA

TAC TGC CTG CAT GAT GGC GTA TGC ATG TAC ATC GAA GCT ATG ACG GAC GTA CTA CCG CAT ACG TAC ATG TAG CTT CGA

CTG GAC AAA TAC GCA TGC AAC TGT GTT GTA GGT TAC ATC GAC CTG TTT ATG CGT ACG TTG ACA CAA CAT CCA ATG TAG GGC GAA CGT TGC CAG TAT CGC GAC CTG AAA TGG TGG GAA CCG CTT GCA ACG GTC ATA GCG CTG GAC TTT ACC ACC CTT

CTG CGT-3 '
GAC GCA-5 '

and

5 '-ATG AAG AAA TAT TGG ATC CAA
3'-TAC TTC TTT ATA ACC TAG GTT

ATG AAC TCT GAT TCC GAA TGC CCG CTG TCT CAT GAC GGT TAC TTG AGA CTA AGG CTT ACG GGC GAC AGA GTA CTG- CCA

TAC TGC CTG CAT GAT GGC GTA TGC ATG TAC ATC GAA GCT ATG ACG GAC GTA CTA CCG CAT ACG TAC ATG TAG CTT CGA

CTG GAC AAA TAC GCA TGC AAC TGT GTT GTA GGT TAC ATC GAC CTG TTT ATG CGT ACG TTG ACA CAA CAT CCA ATG TAG

GGC GAA CGT TGC CAG TAT CGC GAC CTG AAA TGG TGG GAA CCG CTT GCA ACG GTC ATA GCG CTG GAC TTT ACC ACC CTT

CTG CGT-3 '
GAC GCA-5 '

and

5'-AAC TCT GAT TCC GAA TGC CCG CTG TCC-3' -TTG AGA CTA AGG CTT ACG GGC GAC AGG-

CAC GAC GGT TAT TGT CTG CAC GAC GGT GTT TGC ATG-GTG CTG CCA ATA ACA GAC GTG CTG CCA CAA ACG TAC-

TAT ATC GAA GAT CTG GAC AAA TAC GCT TGC AAC TGC-ATA TAG CTT CTA GAC CTG TTT ATG CGA ACG TTG ACG-

GTA GTT GGT TAC ATC GGT GAA CGT TGC CAG TAC CGT-CAT CAA CCA ATG TAG CCA CTT GCA ACG GTC ATG GCA-

GAT CTG AAA TGG TGG GAA CTG CGT-3'
CTA GAC TTT ACC ACC CTT GAC GCA-5 '

and

5 '-AAC TCT GAT TCC GAA TGC CCG CTG TCC-3'-TTG AGA CTA AGG CTT ACG GGC GAC AGG-

CAC GAC GGT TAT TGT CTG CAC GAC GGT GTT TGC ATG-GTG CTG CCA ATA ACA GAC GTG CTG CCA CAA ACG TAC-

TAT ATC GAA GCT CTG GAC AAA TAC GCT TGC AAC TGC-ATA TAG CTT CGA GAC CTG TTT ATG CGA ACG TTG ACG-

GTA GTT GGT TAC ATC GGT GAA CGT TGC CAG TAC CGT-CAT CAA CCA ATG TAG CCA CTT GCA ACG GTC ATG GCA-

GAT CTG AAA TGG TGG GAA CCG CCA-3 *
CTA GAC TTT ACC ACC CTT GGC GGT-5 ' .

11. A manufactured gene according to claim

7 wherein the base codons specifying urogastrone are preceded and/or followed by a sequence of bases comprising a portion of a base sequence which provides a recognition site for restriction endonuclease cleavage of a DNA sequence.

12. A fusion gene comprising a manufactured gene according to either of claims 1 to 7 fused to a second gene capable of directing synthesis of a second polypeptide in a manner permitting the synthesis of a fused polypeptide including urogastrone polypeptide or a urogastrone polypeptide analog.

13. A fusion gene according to claim 12 wherein said second gene is a gene directing synthesis of β-galactosidase enzyme.

14. A fusion gene according to claim 12 wherein said second gene is a gene directing synthesis of β-lactamase.

15. A biologically functional DNA microorganism transformation vector including a manufactured gene according to claim 1.

16. A biologically functional DNA icroorgan- ism transformation vector including a manufactured gene according to claim 7.

17. A biologically functional DNA microorganism transformation vector including a fusion gene according to claim 12.

18. A vector according to either of claims 15, 16 or 17 which is a. circular DNA plasmid.

19. A microorganism transformed with a vector according to either of claims 15, 16 or 17.

20. A process for the production of urogastrone polypeptide comprising:
growing, under appropriate nutrient conditions, microorganisms transformed with a biologically functional DNA including a manufactured gene according to claim 1, whereby said microorganisms express said gene and produce urogastrone polypeptide.

21. A process according to claim 20 wherein the microorganisms grown are E. coli microorganisms.

22. A process for the production of urogas-trone polypeptide analog comprising: growing, under appropriate nutrient conditions, microorganisms transformed with a biologically functional DNA including a manufactured gene according
to claim 7, whereby said microorganisms express said
gene and produce urogastrone polypeptide.

23. A process according to claim 22 wherein the microorganisms grown are E. coli microorganisms.

24. A polypeptide product of the expression in a microorganism of a manufactured gene according
to claim 1.

25. A polypeptide product of the expression in a microorganism of a manufactured gene according
to claim 7.

26. A product according to claim 25 which
is [Asp 25] urogastrone.

27. A product according to claim 25 which
is [Pro 52, Pro53] urogastrone.

28. A product according to claim 25 which
is [Met ] urogastrone.

29. A product according to claim 25 which
is [Met"8, Lys*"7, Lys-6, Tyr"5, Trp"4, He"3, Gin"2,
Met ] urogastrone .

30. A reagent material comprising a radio-labelled manufactured gene according to claim 1.

31. A reagent material comprising a radio-labelled polypeptide product of claim 24 or claim
25.

OMPI

32. A reagent material according to claim
125
30 or 31 wherein said radiolabel is I

33. A reagent material comprising a tagged antibody to a polypeptide according to claim 24 or
claim 25, coated on the surface of a plastic bead.

OMPI_