Evolved sortases and uses thereof
Evolved sortases exhibiting enhanced reaction kinetics and/or altered substrate preferences are provided herein, for example evolved sortases that bind recognitions motifs comprising a LAXT or LPXS sequence. Also provided are methods (e.g., orthogonal transpeptidation and diagnostics methods) for using such sortases. Kits comprising materials, reagents, and cells for carrying out the methods described herein are also provided.
1. A Staphylococcus aureus ( S. aureus ) Sortase A variant that binds substrates comprising the sequence LAXT, wherein X represents any amino acid, and wherein the S. aureus Sortase A variant comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of S. aureus Sortase A as provided as SEQ ID NO: 1, and wherein the amino acid sequence of the Sortase A variant includes a mutation selected from the group consisting of K84R, R99H, R99K, S102C, A104H, E105D, K138I, K138V, K138P, K145E, K152I, D160K, K162R, K162H, T164N, V168I, K177G, K177R, I182F, and K196S.
2. The Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 1 , wherein the S. aureus Sortase A variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence provided in SEQ ID NO: 1.
3. The Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 1 , wherein the amino acid sequence of the S. aureus Sortase A variant comprises at least two mutations, as compared to the amino acid sequence of S. aureus Sortase A provided as SEQ ID NO: 1.
4. The Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 1 , wherein the substrate comprises the amino acid sequence LAXTX, wherein each occurrence of X independently represents any amino acid.
5. The Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 1 , wherein the substrate comprises the amino acid sequence LAETG (SEQ ID NO: 5).
6. A Staphylococcus aureus ( S. aureus ) Sortase A variant that binds substrates comprising the sequence LPXS, wherein X represents any amino acid, the S. aureus Sortase A variant comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of S. aureus Sortase A as provided as SEQ ID NO: 1, wherein the amino acid sequence of the Sortase A variant includes a mutation selected from the group consisting of N98D, S102C, A104V, A118S, A118T, F122A, K134G, K134P, E189V, E189F, and E189P.
7. A method for transpeptidation, the method comprising contacting the S. aureus Sortase A variant of claim 1 with a substrate comprising an LAXT amino acid sequence, wherein X represents any amino acid, and a substrate comprising a GGG sequence under conditions suitable for sortase-mediated transpeptidation.
8. A method for transpeptidation comprising contacting the Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 6 with a substrate comprising an LPXS amino acid sequence, wherein X represents any amino acid, and a substrate comprising a GGG sequence under conditions suitable for sortase-mediated transpeptidation.
9. A method for N-terminal protein modification comprising contacting a protein comprising a N-terminal GGG sequence with the Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 1 , and a sortase substrate comprising a LAXT sequence, respectively, under conditions suitable for sortase-mediated transpeptidation, wherein X represents any amino acid.
10. A method for C-terminal protein modification comprising contacting a protein comprising a C-terminal LAXT sequence with the Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 1 , respectively, and a sortase substrate comprising a GGG sequence under conditions suitable for sortase-mediated transpeptidation, wherein X represents any amino acid.
11. A method for modifying a protein comprising a sortase recognition motif in a cell or tissue, the method comprising contacting the protein with the Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 1 and a sortase substrate comprising a sortase recognition motif under conditions suitable for sortase-mediated transpeptidation, wherein
(a) the protein comprises a N-terminal sortase recognition motif, and the sortase substrate comprises a C-terminal sortase recognition motif; or
(b) the protein comprises a C-terminal sortase recognition motif, and the sortase substrate comprises a N-terminal sortase recognition motif,
wherein the N-terminal sortase recognition motif comprises the sequence GGG, and the C-terminal sortase recognition motif comprises the sequence LAXT, wherein X represents any amino acid.
12. The Staphylococcus aureus ( S. aureus ) sortase A variant of claim 1 , wherein the amino acid sequence of the Sortase A variant includes two mutations selected from the group consisting of K84R, R99H, R99K, S102C, A104H, E105D, K138I, K138V, K138P, K145E, K152I, D160K, K162R, K162H, T164N, V168I, K177G, K177R, I182F, and K196S.
13. The Staphylococcus aureus ( S. aureus ) sortase A variant of claim 1 , wherein the amino acid sequence of the Sortase A variant includes three mutations selected from the group consisting of K84R, R99H, R99K, S102C, A104H, E105D, K138I, K138V, K138P, K145E, K152I, D160K, K162R, K162H, T164N, V168I, K177G, K177R, I182F, and K196S.
14. The Staphylococcus aureus ( S. aureus ) sortase A variant of claim 1 , wherein the amino acid sequence of the Sortase A variant includes four mutations selected from the group consisting of K84R, R99H, R99K, S102C, A104H, E105D, K138I, K138V, K138P, K145E, K152I, D160K, K162R, K162H, T164N, V168I, K177G, K177R, I182F, and K196S.
15. The Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 1 , wherein the S. aureus Sortase A variant comprises an amino acid sequence that is at least 98% identical to the amino acid sequence provided in SEQ ID NO: 1.
16. The Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 1 , wherein the S. aureus Sortase A variant exhibits a ratio of k cat /K M for a substrate comprising the amino acid sequence LAETG (SEQ ID NO: 5) that is least 60-fold greater than the K cat /K M ratio the sortase exhibits for a substrate comprising the amino acid sequence LPETG (SEQ ID NO: 4).
17. The Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 1 , wherein the S. aureus Sortase A variant exhibits a ratio of k cat /K M for a substrate comprising the amino acid sequence LAETG (SEQ ID NO: 5) that is least 100-fold greater than the K cat /K M ratio the sortase exhibits for a substrate comprising the amino acid sequence LPETG (SEQ ID NO: 4).
18. The Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 1 , wherein the S. aureus Sortase A variant exhibits a ratio of k cat /K M for a substrate comprising the amino acid sequence LAETG (SEQ ID NO: 5) that is least 140-fold greater than the K cat /K M ratio the sortase exhibits for a substrate comprising the amino acid sequence LPETG (SEQ ID NO: 4).
19. The Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 1 , wherein the S. aureus Sortase A variant exhibits a K M for a substrate comprising the amino acid sequence LAETG (SEQ ID NO: 5) that is at least 15-fold less than the K M for substrates comprising the amino acid sequence LPETG (SEQ ID NO: 4).
20. The Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 1 , wherein the S. aureus Sortase A variant exhibits a K M for a substrate comprising the amino acid sequence LAETG (SEQ ID NO: 5) that is at least 20-fold less than the K M for substrates comprising the amino acid sequence LPETG (SEQ ID NO: 4).
21. The Staphylococcus aureus ( S. aureus ) sortase A variant of claim 6 , wherein the amino acid sequence of the Sortase A variant includes two mutations selected from the group consisting of N98D, S102C, A104V, A118S, A118T, F122A, K134G, K134P, E189V, E189F, and E189P.
22. The Staphylococcus aureus ( S. aureus ) sortase A variant of claim 6 , wherein the amino acid sequence of the Sortase A variant includes three mutations selected from the group consisting of N98D, S102C, A104V, A118S, A118T, F122A, K134G, K134P, E189V, E189F, and E189P.
23. The Staphylococcus aureus ( S. aureus ) sortase A variant of claim 6 , wherein the amino acid sequence of the Sortase A variant includes four mutations selected from the group consisting of N98D, S102C, A104V, A118S, A118T, F122A, K134G, K134P, E189V, E189F, and E189P.
24. The Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 6 , wherein the S. aureus Sortase A variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence provided in SEQ ID NO: 1.
25. The Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 6 , wherein the S. aureus Sortase A variant comprises an amino acid sequence that is at least 98% identical to the amino acid sequence provided in SEQ ID NO: 1.
26. A method for N-terminal protein modification comprising contacting a protein comprising a N-terminal GGG sequence with a the Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 6 , and a sortase substrate comprising a LPXS sequence, respectively, under conditions suitable for sortase-mediated transpeptidation, wherein X represents any amino acid.
27. A method for C-terminal protein modification comprising contacting a protein comprising a C-terminal LPXS sequence with a the Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 6 , respectively, and a sortase substrate comprising a GGG sequence under conditions suitable for sortase-mediated transpeptidation, wherein X represents any amino acid.
28. A method for modifying a protein comprising a sortase recognition motif in a cell or tissue, the method comprising contacting the protein with the Staphylococcus aureus ( S. aureus ) Sortase A variant of claim 6 and a sortase substrate comprising a sortase recognition motif under conditions suitable for sortase-mediated transpeptidation, wherein
(a) the protein comprises a N-terminal sortase recognition motif, and the sortase substrate comprises a C-terminal sortase recognition motif; or
(b) the protein comprises a C-terminal sortase recognition motif, and the sortase substrate comprises a N-terminal sortase recognition motif,
wherein the N-terminal sortase recognition motif comprises the sequence GGG, and the C-terminal sortase recognition motif comprises the sequence LPXS, wherein X represents any amino acid.