Fully active alternansucrases partially deleted in its carboxy-terminal and amino-terminal domains and mutants thereof

• US 20060127328A1
• Filed: 12/14/2004
• Published: 06/15/2006
• Est. Priority Date: 12/14/2004
• Status: Active Grant

• Current Assignee: Centre National De La Recherche Scientifique
• Original Assignee: Centre National De La Recherche Scientifique
• Inventors: Remaud-Simeon, Magali, Monsan, Pierre, Joucla, Gilles
• Granted Patent: US 7,524,645 B2
• Time in Patent Office: Days
• US Class Current: 424/50
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• CPC Class Codes: C12Y 204/0114 Alternansucrase (2.4.1.140)

Abstract

Nucleic acid sequences of truncated or mutated alternansucrases, vectors containing these nucleic acids sequences, host cells transformed with the nucleic acid sequences encoding truncated or mutated alternansucrases are provided. Furthermore, a process to recombinantly alternansucrase with a high level of expression, while retaining the enzymatic activity is described.

Claims

1. A nucleotide sequence consisting essentially of a nucleotide sequence in FIG. 1 (SEQ ID NO. 1), starting from a nucleotide at position 195 to the nucleotide at position 4241 (SEQ ID No. 2) or 4469 (SEQ ID No. 3) or a nucleotide sequence starting from a nucleotide at position 1218 to the nucleotide at position 4469 (SEQ ID No. 4) of SEQ ID No.1, a sequence complementary to SEQ ID Nos. 2 or 3 or 4 or a sequence that hybridizes to SEQ ID Nos. 2 or 3 or 4 under stringent hybridization conditions, provided that alternansucrase enzyme activity is retained.

2. The nucleotide sequence according to claim 1, wherein the nucleotides at positions 2220 to 2222 can be selected from AGT (SEQ ID No. 5), AGC (SEQ ID No. 6), TCT (SEQ ID No. 7), TCC (SEQ ID No. 8), TCA (SEQ ID No. 9), TCG (SEQ ID No. 10) and the nucleotides at positions 2226 to 2228 can be selected from MT (SEQ ID No. 11) or AAC (SEQ ID No. 12) wherein T can be replaced by U, a sequence complementary to SEQ ID Nos. 5 to 12 or a sequence that hybridizes to SEQ ID Nos. 5 to 12 under stringent hybridization conditions provided that alternansucrase enzyme activity is retained.

3. The nucleotide sequence according to claim 1, wherein the nucleotides at positions 2496 to 2498 are selected from AGT (SEQ ID No. 13), AGC (SEQ ID No. 14), TCT (SEQ ID No. 15), TCC (SEQ ID No. 16), TCA (SEQ ID No. 17) or TCG (SEQ ID No. 18) and the nucleotides at positions 2499 to 2501 are selected from GAA (SEQ ID No. 19) or GAG (SEQ ID No. 20) and the nucleotides at positions 2502 to 2504 are selected from GTT (SEQ ID No. 21), GTC (SEQ ID No. 22), GTA (SEQ ID No. 23) and GTG (SEQ ID No. 24) wherein T can be replaced by U, a sequence complementary to SEQ ID Nos. 13 to 24 or a sequence that hybridizes to SEQ ID Nos. 13 to 24 under stringent hybridization conditions provided that alternansucrase enzyme activity is retained.

4. The nucleotide sequence according to claim 1, wherein said nucleotide sequence has at least 70%, or 80% or 90% sequence identity to that of SEQ ID. Nos. 2 or SEQ ID No. 3 or SEQ ID No. 4.

5. The nucleotide sequence according to claim 2, wherein said nucleotide sequence has at least 70%, or 80% or 90% sequence identity to that of any one of SEQ ID. Nos. 5 to 12.

6. The nucleotide sequence according to claim 3, wherein said nucleotide sequence has at least 70%, or 80% or 90% sequence identity to that of SEQ ID. Nos. 13 or SEQ ID No. 24.

7. A vector containing the nucleic acid sequence according to any one of claims 1 to 6.

8. The vector according to claim 7, which is a plasmid.

9. The vector according to claim 8, selected from the group of [pBad asr C-del bis], [pBad asr C-del bis Δ

Thio], [pBad asr core 1425] and [pBad asr C-del 1349].

10. Host cells transformed with the vector according to claim 7.

17. A method for the preparation of a mutated or truncated alternansucrase, said method comprising:

(a) culturing host cells of claim 10 under conditions permitting the expression of an alternansucrase; and

(b) isolating said alternansucrase from the culture medium.

18. The method according to claim 17, further comprising the step of purifying the isolated alternansucrase.

19. The method according to claim 17, further comprising the step of treating urea and renaturing an insoluble alternansucrase.

11. A protein encoded by the nucleotide sequence of SEQ ID Nos. 2 to 24.

12. A truncated or a mutated alternansucrase, which retains enzymatic activity.

13. The truncated or a mutated alternansucrase according to claim 12, which consists essentially of SEQ ID Nos. 26 to 30.

14. A fusion protein comprising a protein tag at the N- or C-terminal of the amino acid sequences of SEQ ID Nos. 26 to 30.

15. The fusion protein according to claim 14, wherein the protein tag is a thioredoxin situated at the N-terminal.

16. The fusion protein according to claim 14, wherein the protein tag is a histidine situated at the C-terminal.

20. A method to produce alternans or oligoalternans, said method comprising;

reacting the truncated or mutated alternansucrase of SEQ ID Nos, 26 to 30 with maltose thereby producing alternans or oligoalternans.

21. A composition, said composition comprising a truncated or mutated alternansucrase of SEQ ID Nos. 26 to 30 and a pharmaceutically acceptable vehicle.

22. The composition according to claim 21, wherein the pharmaceutically acceptable vehicle is selected from saline and adjuvants.

23. A process to reduce dental caries, said process comprising administering to mammals in need of such treatment a pharmaceutically acceptable amount of the mutated or truncated alternansucrase of SEQ ID Nos. 26 to 30.