pH-insensitive glucose indicator protein
View Patent ↗The present invention encompasses a glucose indicator protein, a biosensor comprising one or more glucose indicator proteins, and methods of use thereof.
1. A pH insensitive glucose indicator protein (GIP) that has a glucose binding dissociation constant (K d ) between about 1 mM and about 20 mM comprising:
a. a glucose binding protein that changes conformation upon exposure to glucose, wherein said glucose binding protein comprises amino acids 24-333 of SEQ ID NO: 1 and has a threonine substituted for phenylalanine at position 39;
b. at least one pH insensitive fluorescence donor coupled to the glucose binding protein; and
c. at least one pH insensitive fluorescence acceptor coupled to the glucose binding protein, wherein the conformational changes of the glucose binding protein upon binding or releasing glucose produces a detectable and reversible signal change.
2. The GIP of claim 1 , wherein the donor fluorescent protein moiety and the acceptor fluorescent protein moiety are Aequorea-related fluorescent protein moieties.
3. The GIP of claim 1 , wherein the donor is enhanced cyan fluorescent protein (ECFP) and the acceptor is yellow fluorescent protein (YFP).
4. The GIP of claim 1 , wherein the donor is fused to the N terminus of the glucose binding protein and the acceptor is fused to the C terminus of the glucose binding protein.
5. The GIP of claim 1 , further comprising two donors.
6. A pH insensitive glucose indicator protein (GIP) that produces a detectable signal in the presence of from at least 1 mM to about 32 mM glucose comprising:
(a) a glucose binding protein that changes conformation upon exposure to glucose, wherein said glucose binding protein comprises amino acids 24-333 of SEQ ID NO: 1 and has a threonine substituted for phenylalanine at position 39;
(b) at least one pH insensitive fluorescence donor coupled to the glucose binding protein; and
(c) at least one pH insensitive fluorescence acceptor coupled to the glucose binding protein, wherein the conformational changes of the glucose binding protein upon binding or releasing glucose produces a detectable and reversible signal change.
7. The GIP of claim 6 , wherein the donor fluorescent protein moiety and the acceptor fluorescent protein moiety are Aequorea-related fluorescent protein moieties.
8. The GIP of claim 6 , wherein the donor is enhanced cyan fluorescent protein (ECFP) and the acceptor is yellow fluorescent protein (YFP).
9. The GIP of claim 6 , wherein the donor is fused to the N terminus of the glucose binding protein and the acceptor is fused to the C terminus of the glucose binding protein.
10. The GIP of claim 6 , further comprising two donors.