DNA sequences encoding various allelic forms of mature thaumatin, recombinant plasmids comprising said DNA's and a process for their preparation, bacterial cultures comprising said recombinant plasmids, and method for producing mature thaumatin

Claim 2

DNA sequences encoding the various allelic forms of mature thaumatin according to Figures 1 and 2.

Claim 3

2 . A recombinant plasmid comprising the DNA sequences coding for the various allelic forms of matured thaumatin and specific DNA sequences which regulate the expression of the structural genes, consisting of an inducible or constitutive regulon.

Claim 4

2 . A recombinant plasmid comprising the DNA sequences coding for the various allelic forms of matured thaumatin and specific DNA sequences which regulate the expression of the structural genes, consisting of an inducible or constitutive regulon.

Claim 5

A recombinant plasmid according to claim 2, in which the specific DNA sequences which regulate the expression of the structural genes comprise an inducible regulon consisting of a double lac UV5 system; plasmid pUR 520 according to Figure 8.

Claim 6

A recombinant plasmid according to claim 2, in which the specific DNA sequences which regulate the expression of the structural genes comprise a modified tryptophan system; plasmid pUR 530 according to Figure 8.

Claim 7

A recombinant plasmid according to claim 2, in which the specific DNA sequences which regulate the expression of the structural genes comprise a modified M13 gene VIII system; plasmid pUR 540 according to Figure 8.

Claim 8

A recombinant plasmid sccording to any one of the preceding claims 1-5, in which the structural gene is linked to the EcoRI site of the regulon through an - EcoRI-treated DNA linker having the nucleotide sequence (5')pCAT(N) n GAATTC(N') n ATG OH (3'), wherein n = 0, 1, 2 or 3, and

Claim 9

N and N' are any of the nucleotides A, T, G or C, with proviso that in the double-stranded structure N and N' are such that a rotational symmetrical structure is present.

Claim 10

N and N' are any of the nucleotides A, T, G or C, with proviso that in the double-stranded structure N and N' are such that a rotational symmetrical structure is present.

Claim 11

DNA-linkers having the nucleotide sequences (5') pCAT(N) n GAATTC(N') n ATG OH (3'), wherein n = 0, 1, 2 or 3, and N and N' are any of the nucleotides A, T, G or C, with the proviso that N and N' are such that in the double-stranded structure a rotational symmetrical structure is present.

Claim 12

DNA-linkers having the nucleotide sequences (5') pCAT(N) n GAATTC(N') n ATG OH (3'), wherein n = 0, 1, 2 or 3, and N and N' are any of the nucleotides A, T, G or C, with the proviso that N and N' are such that in the double-stranded structure a rotational symmetrical structure is present.

Claim 13

A bacterial culture cloned from at least one microorganism comprising plasmids as claimed in any one of the preceding claims.

Claim 14

A bacterial culture comprising E.coli cells containing pUR 520 with or without the AATT sequence in the linker between the regulon and the thaumatin gene.

Claim 15

A bacterial culture comprising E.coli cells containing pUR 520 with or without the AATT sequence in the linker between the regulon and the thaumatin gene.

Claim 16

A bacterial culture comprising E.coli cells containing pUR 530 with or without the AATT sequence in the linker between the regulon and the thaumatin gene.

Claim 17

A bacterial culture comprising E.coli cells containing pUR 530 with or without the AATT sequence in the linker between the regulon and the thaumatin gene.

Claim 18

A bacterial culture comprising E.coli cells containing pUR 540 with or without the AATT sequence in the linker between the regulon and the thaumatin gene.

Claim 19

A bacterial culture comprising E.coli cells containing pUR 540 with or without the AATT sequence in the linker between the regulon and the thaumatin gene.

Claim 20

A process for producing thaumatin which comprises incorporating the plasmids claimed in any one of the preceding claims in a microbial cloning vehicle, cultivating the transformed cells and isolating the thaumatin produced by said cells.

Claim 21

A process for producing DNA sequences encoding the various allelic forms of mature thaumatin, characterized in that: (1) thaumatin messenger RNA-containing material is isolated from arils of Thaumatococcus daniellii; (2) said thaumatin messenger RNA-containing material is purified; (3) the thaumatin messenger RNA is converted into single-stranded DNA using a reverse transcriptase and subsequently to double-stranded DNA by a DNA polymerase; (4) said double-stranded DNA is integrated in plasmids; (5) said plasmids are transferred into bacterial cells and hybrid plasmid DNA molecules are selected to yield a plasmid containing a substantially complete copy of thaumatin mRNA; (6) Single-stranded DNA is produced and used as template for complementary DNA synthesis in the presence of a primer; (7) denaturation of double-stranded DNA is carried out and the complementary DNA is used as template in the presence of a primer to obtain a double-stranded DNA; (8) said double-stranded DNA is treated with an appropriate enzyme to obtain DNA sequences encoding at least one of the various allelic forms of mature thaumatin according to Figures 1 and 2. 2. A process for producing recombinant plasmids, characterized in that a recombinant plasmid is produced which comprises DNA sequences encoding the various allelic forms of mature thaumatin according to Figures 1 and 2 and specific DNA sequences which regulate the expression of the structural genes, consisting of an inducible or constitutive regulon. 3. A process according to claim 2, characterized in that a plasmid pUR 520 according to Figure 8 is produced in which the specific DNA sequences which regulate the expression of the structural genes comprise an inducible regulon consisting of a double lac UV5 system. 4. A process according to claim 2, characterized in that a plasmid pUR 530 according to Figure 8 is produced in which the specific DNA sequences which regulate the expression of the structural genes comprise a modified tryptophan system. 5. A process according to claim 2, characterized in that a plasmid pUR 540 according to Figure 8 is produced in which the specific DNA sequences which regulate the expression of the structural genes comprise a modified M13 gene VIII system. 6. A process according to any one of the preceding claims 2 - 5, characterized in that the structural gene is linked to the EcoRI site of the regulon through an EcoRI- treated DNA linker having the nucleotide sequence (S')pCAT(N)nGAATTC(N')nATGOH (3'), wherein n = 0, 1, 2 or 3, and N and N' are any of the nucleotides A, T, G or C, with the proviso that in the double-stranded structure N and N' are such that a rotational symmetrical structure is present.

Claim 22

A process for producing thaumatin, characterised in that . (1) plasmids produced according to the process claimed in any one of the preceding claims 2 - 6 are incorporated in microbial cloning vehicles; (2) transformed cells are cultivated; and (3) thaumatin produced by said cells is isolated.

Claim 23

A process according to claim 7, characterized in that pUR 520, pUR 530 and/or pUR 540 with or without the AAT T sequence in the linker between the regulon and the thaumatin gene are incorporated in E-coli cells.