Split inteins with exceptional splicing activity

1. A split intein N-fragment comprising an amino acid sequence of at least 98% sequence identity to

(SEQ ID NO: 1)

CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNR

GEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGL

or to

(SEQ ID NO: 2)

CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNR

GEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGL

P.

2. A complex comprising the split intein N-fragment of claim 1 and a compound.

3. The complex of claim 2 , wherein the compound is selected from the group consisting of (i) a peptide or a polypeptide, (ii) an antibody chain, (iii) an antibody heavy chain and (iv) a compound comprising a peptide, an oligonucleotide, a drug or a cytotoxic molecule.

4. A split intein C-fragment comprising an amino acid sequence of at least 98% sequence identity to

(SEQ ID NO: 3)

VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN,

to

(SEQ ID NO: 4)

MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN

or to

(SEQ ID NO: 389)

VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN.

5. A complex comprising the split intein C-fragment of claim 4 and a compound.

6. The complex of claim 5 , wherein the compound is selected from the group consisting of:

(i) a peptide or a polypeptide,

(ii) a compound comprising a peptide, an oligonucleotide, a drug, or a cytotoxic molecule,

(iii) a 1,2-amino thiol bonded to a peptide, an oligonucleotide, a drug, or a cytotoxic molecule,

(iv) a 1,2-amino alcohol bonded to a peptide, an oligonucleotide, a drug, or a cytotoxic molecule, and

(v) a dendrimer.

7. The complex of claim 5 , wherein the compound is a dendrimer having the structure

wherein R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen (H) and cargo molecules.

8. The complex of claim 7 , wherein R1, R2, R3, and R4 are each a dye molecule or wherein R1, R2, R3, and R4 are each a fluorescein derivative having the structure

9. A complex selected from the group consisting of:

(i) a complex of the structure

wherein IntC is the split intein C-fragment of claim 4 , and

wherein n is from 0 to 8,

(ii) a complex of the structure

wherein IntC is the split intein C-fragment of claim 4 , and

wherein n is from 0 to 8, and

(iii) a complex of the structure

wherein IntC is the split intein C-fragment of claim 4 , and

wherein X is sulfur (S) or oxygen (O).

10. A composition comprising:

the split intein N-fragment of claim 1 ; and

a split intein C-fragment split intein C-fragment comprising:

an amino acid sequence of at least 98% sequence identity to

(SEQ ID NO: 3)

VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN,

(SEQ ID NO: 4)

MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN,

or

(SEQ ID NO: 389)

VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN.

11. A nucleotide plasmid comprising a nucleotide sequence encoding the split intein N-fragment of claim 1 .

12. A nucleotide plasmid comprising a nucleotide sequence encoding the split intein C-fragment of claim 4 .

13. A method for splicing two complexes comprising:

contacting a first complex comprising a first compound and the split intein N-fragment of claim 1 and a second complex comprising a second compound and a split intein C-fragment,

wherein the split intein C-fragment comprises an amino acid sequence of at least 98% sequence identity to

(SEQ ID NO: 3)

VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN,

(SEQ ID NO: 4)

MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN,

or

(SEQ ID NO: 389)

VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN,

wherein the contacting is performed under conditions that permit binding of the split intein N-fragment to the split intein C-fragment to form an intein intermediate; and

reacting the intein intermediate to form a conjugate of the first compound with the second compound.

14. A method selected from the group consisting of:

(i) a method comprising:

contacting a first complex comprising a first compound and the split intein N-fragment of claim 1 and a second complex comprising a second compound and a split intein C-fragment,

wherein the contacting is performed under conditions that permit binding of the split intein N-fragment to the split intein C-fragment to form an intein intermediate; and

reacting the intein intermediate with a nucleophile to form a conjugate of the first compound with the nucleophile; and

(ii) a method comprising:

fusing a first nucleotide sequence encoding an amino acid sequence of the split intein N-fragment of claim 1 with a second nucleotide sequence encoding an amino acid sequence of a split intein C-fragment,

so that the fusion of the first nucleotide sequence and the second nucleotide sequence encodes for a contiguous intein;

wherein the split intein C-fragment comprises an amino acid sequence of at least 98%, sequence identity to

(SEQ ID NO: 3)

VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN,

(SEQ ID NO: 4)

MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN,

or

(SEQ ID NO: 389)

VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN.

15. The method of claim 14 , wherein;

the first compound is a polypeptide or an antibody, or

the second compound is a dendrimer or a polypeptide.

16. An intein comprises an amino acid sequence of at least 98% sequence identity to

(SEQ ID NO: 390)

CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRG

EQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLPV

KIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN.

17. A kit for splicing two complexes together comprising:

the split intein N-fragment of claim 1 ;

a split intein C-fragment comprises an amino acid sequence of at least 98% sequence identity to

(SEQ ID NO: 3)

VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN,

(SEQ ID NO: 4)

MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN,

or

(SEQ ID NO: 389)

VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN;

a reagent for binding the split intein N-fragment to the split intein C-fragment to form an intein intermediate; and

a nucleophilic agent.

18. A gene fusion comprising:

a first nucleotide sequence encoding an amino acid sequence of the split intein N-fragment of claim 1 ;

fused with a second nucleotide sequence encoding a split intein C-fragment comprising an amino acid sequence of at least 98% sequence identity to VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 3), MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 4), or VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN (SEQ ID NO: 389).

19. The gene fusion of claim 18 , comprising:

a first nucleotide sequence encoding the amino acid sequence of a split intein N-fragment comprising

(SEQ ID NO: 1)

CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRG

EQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGL,

fused with a second nucleotide sequence encoding an amino acid sequence of a split intein C-fragment comprising

(SEQ ID NO: 3)

VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN.

20. A polynucleotide encoding the split intein N-fragment of claim 1 .

21. A polynucleotide encoding the split intein C-fragment of claim 4 .