Designer ubiquitin ligases having a non-cleavable SNAP25 domain and E3-ligase domain

1. An isolated recombinant ubiquitin ligase molecule that comprises:

a. a toxin binding domain that has an affinity for an enzymatically active fragment of one or more toxins or toxin serotypes, wherein the toxin binding domain comprises a non-cleavable SNAP-25 domain that is encoded by a nucleic acid molecule that consists essentially of a nucleic acid sequence selected from the group consisting of:

i. a nucleic acid sequence having greater than or equal to about 90% identity with nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

ii. a nucleic acid sequence complement having greater than or equal to about 90% identity with nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

iii. a nucleic acid molecule that hybridizes to nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. a nucleic acid sequence that encodes an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 1 to 206 of SEQ ID NO: 12 or 14; and

b. an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated ubiquitination of the toxin enzymatically active fragment, wherein the E3-ligase domain is encoded by a nucleic acid molecule that consists essentially of a nucleic acid sequence selected from the group consisting of:

i. a nucleic acid sequence having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

ii. a nucleic acid sequence complement having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

iii. a nucleic acid molecule that hybridizes to nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. a nucleic acid sequence that encodes an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 207 to 343 of SEQ ID NO: 12 or 14.

2. The isolated recombinant ubiquitin ligase molecule of claim 1 , wherein the non-cleavable SNAP-25 domain binds to the enzymatically active fragment of a botulinum neurotoxin (BoNT).

3. An isolated recombinant ubiquitin ligase molecule that comprises:

a. a toxin binding domain that has an affinity for an enzymatically active fragment of one or more toxins or toxin serotypes, wherein the toxin binding domain comprises a non-cleavable SNAP-25 domain, wherein the non-cleavable SNAP-25 domain consists essentially of an amino acid sequence selected from the group consisting of:

i. an amino acid sequence encoded by a nucleic acid having greater than or equal to about 90% identity with nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

ii. an amino acid sequence encoded by a complement having greater than or equal to about 90% identity with of nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

iii. an amino acid sequence encoded by a nucleic acid molecule that hybridizes to nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 1 to 206 of SEQ ID NO: 12 or 14; and

b. an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated ubiquitination of the toxin enzymatically active fragment, wherein the E3-ligase domain consists essentially of an amino acid sequence selected from the group consisting of:

i. an amino acid sequence encoded by a nucleic acid having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

ii. an amino acid sequence encoded by a complement having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

iii. an amino acid sequence encoded by a nucleic acid molecule that hybridizes to nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 207 to 343 of SEQ ID NO: 12 or 14.

4. The isolated recombinant ubiquitin ligase molecule of claim 2 , wherein the serotypes comprise BoNT serotypes A, C, or E.

5. An isolated recombinant ubiquitin ligase molecule that is encoded by a nucleic acid molecule that consists essentially of a nucleic acid sequence selected from the group consisting of:

a. a nucleic acid sequence having greater than or equal to about 90% identity with SEQ ID NO: 11 or 13;

b. a nucleic acid sequence complement having greater than or equal to about 90% identity with of SEQ ID NO: 11 or 13;

c. a nucleic acid molecule that hybridizes to SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

d. a nucleic acid sequence that encodes an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in SEQ ID NO: 12 or 14 wherein the isolated recombinant ubiquitin ligase molecule comprises a non-cleavable SNAP-25 toxin binding domain that binds to the enzymatically active fragment of BoNT, and a RING E3-ligase domain that facilitates E2-mediated ubiquitination of the toxin enzymatically active fragment.

6. An isolated recombinant ubiquitin ligase molecule that consists essentially of an amino acid sequence selected from the group consisting of:

a. an amino acid sequence encoded by a nucleic acid having greater than or equal to about 90% identity with SEQ ID NO: 11 or 13;

b. an amino acid sequence encoded by a complement having greater than or equal to about 90% identity with of SEQ ID NO: 11 or 13;

c. an amino acid sequence encoded by a nucleic acid that hybridizes to SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

d. an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in SEQ ID NO: 12 or 14

wherein the isolated recombinant ubiquitin ligase molecule comprises a non-cleavable SNAP-25toxin binding domain that binds to the enzymatically active fragment of BoNT, and a RING E3-ligase domain that facilitates E2-mediated ubiquitination of the toxin enzymatically active fragment.

7. An isolated polypeptide molecule that comprises:

a. a non-cleavable SNAP-25 domain that has affinity to the enzymatically active fragment of a BoNT, wherein the non-cleavable SNAP-25 domain is encoded by a nucleic acid molecule that consists essentially of a nucleic acid sequence selected from the group consisting of:

i. a nucleic acid sequence having greater than or equal to about 90% identity with nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

ii. a nucleic acid sequence complement having greater than or equal to about 90% identity with nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

iii. a nucleic acid molecule that hybridizes to nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. a nucleic acid sequence that encodes an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 1 to 206 of SEQ ID NO: 12 or 14;

b. a BoNT holotoxin, wherein the BoNT holotoxin comprises a deletion in the light chain, a mutation of the heavy chain, or both, wherein the deletion, mutation or both renders BoNT holotoxin atoxic; and

c. an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated degradation of the toxin enzymatically active fragment of a BoNT; wherein the E3-ligase domain is encoded by a nucleic acid molecule that consists essentially of a nucleic acid sequence selected from the group consisting of:

i. a nucleic acid sequence having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

ii. a nucleic acid sequence complement having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

iii. a nucleic acid molecule that hybridizes to nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. a nucleic acid sequence that encodes an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 207 to 343 of SEQ ID NO: 12 or 14;

wherein non-cleavable SNAP-25 domain and the BoNT atoxic holotoxin are fused.

8. The isolated polypeptide molecule of claim 7 , wherein the non-cleavable SNAP-25 domain is fused to the N-terminus of the BoNT atoxic holotoxin.

9. A fusion protein comprising the isolated polypeptide of claim 7 .

10. An isolated recombinant ubiquitin ligase molecule that comprises:

a. a non-cleavable SNAP-25 domain that has affinity to the enzymatically active fragment of a BoNT, wherein the non-cleavable SNAP-25 domain is encoded by a nucleic acid molecule that consists essentially of a nucleic acid sequence selected from the group consisting of:

i. a nucleic acid sequence having greater than or equal to about 90% identity with nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

ii. a nucleic acid sequence complement having greater than or equal to about 90% identity with nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

iii. a nucleic acid molecule that hybridizes to nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. a nucleic acid sequence that encodes an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 1 to 206 of SEQ ID NO: 12 or 14;

b. an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated ubiquitination of the toxin enzymatically active fragment, wherein the E3-ligase domain is encoded by a nucleic acid molecule that consists essentially of a nucleic acid sequence selected from the group consisting of:

i. a nucleic acid sequence having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

ii. a nucleic acid sequence complement having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

iii. a nucleic acid molecule that hybridizes to nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. a nucleic acid sequence that encodes an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 207 to 343 of SEQ ID NO: 12 or 14; and

c. a translocation cellular binding domain or to the N-terminus of the BoNT atoxic holotoxin.

11. The isolated recombinant ubiquitin ligase molecule of claim 10 , wherein the translocation cellular binding domain binds to the cellular membrane of a cell and delivers the recombinant ubiquitin ligase into the cell.

12. An isolated recombinant ubiquitin ligase molecule that comprises:

a. a non-cleavable SNAP-25 domain that binds to the enzymatically active fragment of a BoNT, wherein the non-cleavable SNAP-25 domain is encoded by a nucleic acid molecule that comprises a nucleic acid sequence selected from the group consisting of:

i. a nucleic acid sequence having nucleic acid bases 1 to 618 of SEQ ID NO: 11;

ii. a nucleic acid sequence complement of nucleic acid bases 1 to 618 of SEQ ID NO: 11;

iii. a nucleic acid molecule that hybridizes to nucleic acid bases 1 to 618 of SEQ ID NO: 11 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. a nucleic acid sequence that encodes an amino acid sequence having a sequence set forth in amino acid bases 1 to 206 of SEQ ID NO: 12; and

b. an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated ubiquitination of the toxin enzymatically active fragment.

13. The isolated recombinant ubiquitin ligase molecule of claim 12 , wherein the E3-ligase domain comprises a RING domain encoded by a nucleic acid molecule that comprises a nucleic acid sequence selected from the group consisting of:

i. a nucleic acid sequence having nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

ii. a nucleic acid sequence complement of nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

iii. a nucleic acid molecule with a molecule that hybridizes to nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. a nucleic acid sequence that encodes an amino acid sequence set forth in amino acid bases 207 to 343 of SEQ ID NO: 12 or 14.

14. An isolated recombinant ubiquitin ligase molecule that comprises:

a. a non-cleavable SNAP-25 domain that binds to the enzymatically active fragment of a BoNT, wherein the non-cleavable SNAP-25 domain that comprises an amino acid sequence selected from the group consisting of:

i. an amino acid sequence encoded by a nucleic acid having nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

ii. an amino acid sequence encoded by a complement nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

iii. an amino acid sequence encoded by a nucleic acid molecule that hybridizes to nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. an amino acid sequence having a sequence set forth in amino acid bases 1 to 206 of SEQ ID NO: 12 or 14; and

b. an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated ubiquitination of the toxin enzymatically active fragment.

15. The isolated recombinant ubiquitin ligase molecule of claim 14 , wherein the E3-ligase domain comprises a RING domain that comprises an amino acid sequence selected from the group consisting of:

i. an amino acid sequence encoded by a nucleic acid having nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

ii. an amino acid sequence encoded by a complement of nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

iii. an amino acid sequence encoded by a nucleic acid molecule that hybridizes to nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. an amino acid sequence set forth in amino acid bases 207 to 343 of SEQ ID NO: 12 or 14.

16. An isolated polypeptide molecule that comprises:

a. a non-cleavable SNAP-25 domain that has affinity to the enzymatically active fragment of a BoNT, wherein the non-cleavable SNAP-25 domain consists essentially of an amino acid sequence selected from the group consisting of:

i. an amino acid sequence encoded by a nucleic acid having greater than or equal to about 90% identity with nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

ii. an amino acid sequence encoded by a complement having greater than or equal to about 90% identity with of nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

iii. an amino acid sequence encoded by a nucleic acid molecule that hybridizes to nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 1 to 206 of SEQ ID NO: 12 or 14;

b. a BoNT holotoxin, wherein the BoNT holotoxin comprises a deletion in the light chain, a mutation of the heavy chain, or both, wherein the deletion, mutation or both renders BoNT holotoxin atoxic; and

c. an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated degradation of the toxin enzymatically active fragment of a BoNT; wherein E3-ligase domain comprises an amino acid sequence selected from the group consisting of:

i. an amino acid sequence encoded by a nucleic acid having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

ii. an amino acid sequence encoded by a complement having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

iii. an amino acid sequence encoded by a nucleic acid molecule that hybridizes to nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 207 to 343 of SEQ ID NO: 12 or 14;

wherein non-cleavable SNAP-25 domain and the BoNT atoxic holotoxin are fused.

17. The isolated polypeptide molecule of claim 16 , wherein the non-cleavable SNAP-25 domain is fused to the N-terminus of the BoNT atoxic holotoxin.

18. A fusion protein comprising the isolated polypeptide of claim 16 .

19. An isolated recombinant ubiquitin ligase molecule that comprises:

a. a non-cleavable SNAP-25 domain that has affinity to the enzymatically active fragment of a BoNT, wherein the non-cleavable SNAP-25 domain consists essentially of an amino acid sequence selected from the group consisting of:

i. an amino acid sequence encoded by a nucleic acid having greater than or equal to about 90% identity with nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

ii. an amino acid sequence encoded by a complement having greater than or equal to about 90% identity with of nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

iii. an amino acid sequence encoded by a nucleic acid molecule that hybridizes to nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 1 to 206 of SEQ ID NO: 12 or 14;

b. an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated ubiquitination of the toxin enzymatically active fragment, wherein E3-ligase domain comprises an amino acid sequence selected from the group consisting of:

i. an amino acid sequence encoded by a nucleic acid having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

ii. an amino acid sequence encoded by a complement having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

iii. an amino acid sequence encoded by a nucleic acid molecule that hybridizes to nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 207 to 343 of SEQ ID NO: 12 or 14;

c. a translocation cellular binding domain or to the N-terminus of the BoNT atoxic holotoxin.

20. The isolated recombinant ubiquitin ligase molecule of claim 19 , wherein the translocation cellular binding domain binds to the cellular membrane of a cell and delivers the recombinant ubiquitin ligase into the cell.

21. An isolated recombinant ubiquitin ligase molecule that comprises:

a. a non-cleavable SNAP-25 domain that binds to the enzymatically active fragment of a BoNT, wherein the non-cleavable SNAP-25 domain is encoded by a nucleic acid molecule that comprises a nucleic acid sequence selected from the group consisting of:

i. a nucleic acid sequence having greater than or equal to about 90% identity with nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

ii. a nucleic acid sequence complement having greater than or equal to about 90% identity with nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

iii. a nucleic acid molecule that hybridizes to nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. a nucleic acid sequence that encodes an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 1 to 206 of SEQ ID NO: 12 or 14; and

b. an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated ubiquitination of the toxin enzymatically active fragment.

22. The isolated recombinant ubiquitin ligase molecule of claim 21 , wherein the E3-ligase domain is encoded by a nucleic acid molecule that comprises a nucleic acid sequence selected from the group consisting of:

i. a nucleic acid sequence having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

ii. a nucleic acid sequence complement having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

iii. a nucleic acid molecule that hybridizes to nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. a nucleic acid sequence that encodes an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 207 to 343 of SEQ ID NO: 12 or 14.

23. An isolated recombinant ubiquitin ligase molecule that comprises:

a. a non-cleavable SNAP-25 domain that binds to the enzymatically active fragment of a BoNT, wherein the non-cleavable SNAP-25 domain that comprises an amino acid sequence selected from the group consisting of:

i. an amino acid sequence encoded by a nucleic acid having greater than or equal to about 90% identity with nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

ii. an amino acid sequence encoded by a complement having greater than or equal to about 90% identity with of nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13;

iii. an amino acid sequence encoded by a nucleic acid molecule that hybridizes to nucleic acid bases 1 to 618 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1× and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 1 to 206 of SEQ ID NO: 12 or 14; and

b. an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated ubiquitination of the toxin enzymatically active fragment.

24. The isolated recombinant ubiquitin ligase molecule of claim 23 , wherein the E3-ligase domain comprises an amino acid sequence selected from the group consisting of:

i. an amino acid sequence encoded by a nucleic acid having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

ii. an amino acid sequence encoded by a complement having greater than or equal to about 90% identity with nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13;

iii. an amino acid sequence encoded by a nucleic acid molecule that hybridizes to nucleic acid bases from 619 to 1029 of SEQ ID NO: 11 or 13 under high stringency conditions, wherein said conditions comprise a temperature range between about 40° C. and about 60° C., an SSC concentration range between about 1 and about 10× and a reaction time range of between about 30 seconds and about 36 hours; and

iv. an amino acid sequence having greater than or equal to about 90% similarity to a sequence set forth in amino acid bases 207 to 343 of SEQ ID NO: 12 or 14.