BRET-BASED MPRO BIOSENSOR WITH AN INCREASED RATE OF CLEAVAGE
Example systems, methods, and apparatus are disclosed herein for a BRET-based Miro biosensor including an mNeonGreen (mNG) reporter protein, a NanoLuc (NLuc) reporter protein, and x repeats of an N-terminal autocleavage peptide sequence of M pro . The x repeats of an N-terminal autocleavage peptide sequence of M pro are located between the mNG reporter protein and the NLuc reporter protein.
1 . A BRET-based M pro biosensor comprising:
an mNeonGreen (mNG) reporter protein;
a NanoLuc (NLuc) reporter protein; and
x repeats of an N-terminal autocleavage peptide sequence of M pro ;
wherein the x repeats of an N-terminal autocleavage peptide sequence of M pro are located between the mNG reporter protein and the NLuc reporter protein.
2 . The BRET-based M pro biosensor of claim 1 , wherein the x repeats of an N-terminal autocleavage peptide sequence of M pro are two.
3 . The BRET-based M pro biosensor of claim 1 , wherein the x repeats of an N-terminal autocleavage peptide sequence of M pro are four.
4 . The BRET-based M pro biosensor of claim 1 , wherein the x repeats of an N-terminal autocleavage peptide sequence of M pro are eight.
5 . The BRET-based M pro biosensor of claim 1 , wherein the N-terminal autocleavage peptide sequence of M pro comprises a peptide sequence AVLQSGFR.
6 . The BRET-based M pro biosensor of claim 1 , wherein the x repeats of an N-terminal autocleavage peptide sequence of M pro are twelve.
7 . The BRET-based M pro biosensor of claim 6 , wherein amino acid residues C-terminal to a critical Gln residue are varied.
8 . The BRET-based M pro biosensor of claim 7 , wherein the twelve repeats of an N-terminal autocleavage peptide sequence of M pro comprises Table 1 Sequence ID Listing No. 5.
9 . A BRET-based M pro biosensor comprising:
an mNeonGreen (mNG) reporter protein;
a NanoLuc (NLuc) reporter protein;
x repeats of an N-terminal autocleavage peptide sequence of M pro ;
a first nanobody; and
a second nanobody;
wherein the x repeats of an N-terminal autocleavage peptide sequence of M pro are located between the mNG reporter protein and the NLuc reporter protein.
10 . The BRET-based M pro biosensor of claim 9 , wherein the first nanobody is NB1D10.
11 . The BRET-based M pro biosensor of claim 10 , wherein the first nanobody is a C-terminal fusion.
12 . The BRET-based M pro biosensor of claim 10 , wherein the first nanobody is an N-terminal fusion.
13 . The BRET-based M pro biosensor of claim 9 , wherein the second nanobody is NB2E3.
14 . The BRET-based M pro biosensor of claim 13 , wherein the second nanobody is a C-terminal fusion.
15 . The BRET-based M pro biosensor of claim 13 , wherein the second nanobody is an N-terminal fusion.
16 . The BRET-based M pro biosensor of claim 9 , wherein the x repeats of an N-terminal autocleavage peptide sequence of M pro are two.
17 . The BRET-based M pro biosensor of claim 9 , wherein the N-terminal autocleavage peptide sequence of M pro comprises a peptide sequence AVLQSGFR.
18 . A method of creating a BRET-based M pro biosensor comprising:
placing x repeats of an N-terminal autocleavage peptide sequence of M pro between an mNeonGreen (mNG) reporter protein and a NanoLuc (NLuc) reporter protein.
19 . The method claim 18 further comprising, inserting a first nanobody between the nNG reporter protein and the x repeats of an N-terminal autocleavage peptide sequence of M pro , and inserting a second nanobody between the x repeats of an N-terminal autocleavage peptide sequence of M pro and the Nluc reporter protein.
20 . The method of claim 18 , wherein the x repeats of an N-terminal autocleavage peptide sequence of M pro are two.