TelA variants, compositions, and methods
The present disclosure relates, according to some embodiments, to telomere resolvases (e.g., TelA variants) having a turnover number over 1.
1. A variant protelomerase capable of cleaving and rejoining a polynucleotide comprising a nucleotide sequence according to SEQ ID NO:16 and having a turnover number≥1.1, wherein the variant protelomerase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:1 and having the following properties:
(a) X1 is T, M-T or M-H 8 -S-G-T;
(b) X97 is K or R;
(c) X98 is A; and
(d) X337 is G or G-H 6 ,
provided that if X1 is M-H 8 -S-G-T, X337 is G and if X337 is G-H 6 , X1 is T or M-T.
2. A variant protelomerase according to claim 1 , wherein the amino acid sequence further has the following additional properties:
(e) Y96 is Y;
(f) R100 is R;
(g) R101 is R; and
(h) K103 is K.
3. A variant protelomerase according to claim 1 , wherein the amino acid sequence has (a) X1 is M-T, (b) X97 is K or R, and (d) X337 is G.
4. A variant protelomerase according to claim 1 , wherein the amino acid sequence has (a) X1 is M-H 8 -S-G-T, (b) X97 is K or R, and (d) X337 is G.
5. A variant protelomerase according to claim 1 , wherein the amino acid sequence has (a) X1 is M-T, (b) X97 is K or R, and (d) X337 is G-H 6 .
6. A variant protelomerase according to claim 1 , wherein the amino acid sequence has at least 97% identity to SEQ ID NO:1.
7. A composition comprising:
a variant protelomerase according to claim 1 ; and
a buffer.
8. A composition according to claim 7 , wherein the buffer is a storage buffer or a reaction buffer.
9. A composition according to claim 7 further comprising a salt, a protein, a stabilizer, a detergent, a polyanion, a polynucleotide, a cell, a biological fluid or secretion, an aptamer, a pH indicator, a crowding agent, a sugar, a starch, cellulose, a glass-forming agent, a lipid, an oil, aqueous media, a support, or any combination thereof.
10. A composition according to claim 9 , wherein the salt comprises NaCl, MgCl 2 , MnCl 2 , CaCl 2 , an amino acid salt or any combination thereof.
11. A composition according to claim 9 , wherein the detergent is selected from an ionic detergent, a non-ionic detergent, and a zwitterionic detergent.
12. A composition according to claim 9 , wherein the polynucleotide is a variant TelA DNA substrate having a nucleotide sequence comprising at least one copy of SEQ ID NO:16.
13. A composition according to claim 7 further comprising propanediol, betaine, CaCl 2 , NaCl, KCl, potassium glutamate, poloxamer, octoxinol-9, polysorbate20, or combinations thereof.
14. A kit comprising:
a variant protelomerase according to claim 1 ; and
a buffer.
15. A kit according to claim 14 , wherein the buffer is a storage buffer or a reaction buffer.
16. A kit according to claim 14 further comprising a salt, a protein, a stabilizer, a detergent, a polyanion, a polynucleotide, a cell, a biological fluid or secretion, an aptamer, a pH indicator, a crowding agent, a sugar, a starch, cellulose, a glass-forming agent, a lipid, an oil, aqueous media, a support, or any combination thereof.
17. A kit according to claim 16 , wherein the salt comprises NaCl, MgCl 2 , MnCl 2 , CaCl 2 , an amino acid salt or any combination thereof.
18. A kit according to claim 16 , wherein the detergent is selected from an ionic detergent, a non-ionic detergent, and a zwitterionic detergent.
19. A kit according to claim 14 , wherein the variant protelomerase and the buffer are in separate containers.
20. A kit according to claim 16 , wherein the polynucleotide is an adapter.
21. A kit according to claim 16 , wherein the polynucleotide is a variant protelomerase DNA substrate having a nucleotide sequence comprising at least one copy of SEQ ID NO:16.