204
4. 8 mM TVMV peptide substrate dissolved in DMSO and stored
at −80 °C.
5. 20 μM TVMV peptide substrate diluted in protease assay buffer
and stored on ice.
Table 1
Summary of working concentration of individual protease switches in different assays and
applications
Protease switch
Concentration
(final assay)
Concentration
(4× Stock)
Target analyte
Type of measurement or
application
TVMV
Thr
-AI
500 nM
2 μM
Thrombin
Maximum induction ratio
HCV
Tvmv
-AI
500 nM
2 μM
TVMV
Maximum induction ratio
TVMV-FN3- PDZ-AI
250 nM
1 μM
Ligand B1
Maximum induction Ratio
TVMV-FN3- PDZ -AI 250 nM
1 μM
Ligand B2
Maximum induction ratio
TVMV-FN3- PDZ -AI 100 nM
400 nM
Ligand B1
K D determination
TVMV-FN3- PDZ -AI 10 nM
40 nM
Ligand B2
K D determination
SH3
Dom
-TVMV- FN3PDZ-AI
10 nM
40 nM
Ligand B1
and B2
Integrated signal sensing
and amplification circuit
SH3
Pep
- HCV
TVMV
- AI
200 nM
800 nM
Ligand B1
and B2
Integrated signal sensing
and amplification circuit
FKBP12- TVMV
Thr
- AI 100 pM
400 pM
Rapamycin
Proximity sensor
HCV
Tvmv
-AI-FRB
200 nM
800 nM
Rapamycin
Proximity sensor
Table 2
Summary of protease substrates, affinity clamp ligands, and AI domains
Protease
Substrate Sequence
Comments
TVMV
ANA- GETVRFQSDT(164)-NH 2 Used in library screening and protease activity assays
HCV
ANA- DDVTPCSMS(164)-NH 2 Used in library screening and protease activity assays
TVMV-DD ANA-GETVRFQSDT(164)
DD-NH 2
Used for measuring enzyme kinetics. Contains
additional Asp residues for improved water
solubility
B1 Ligand
(“Strong”)
NH 2 -RGSIDTWV-COOH
K D = 0.6 nM for the native affinity clamp ePDZ-b1
B2 Ligand
(“Weak”)
NH 2 -PQPVDSWV-COOH
K D = 5 nM for the native affinity clamp ePDZ-b1
AI Domain NH 2 -EYVRFAPGST- COOH
TVMV specific AI domain
ANA refers to a 5-amino-2-nitrobenzoyl group
164 refers to a Mimotopes specific code for lysine coupled to a 7-methoxycoumarinyl-4-acetyl group
Viktor Stein and Kirill Alexandrov