Oxidation reduction sensitive green fluorescent protein variants
First Claim
1. An isolated mutant green fluorescent protein (GFP), with a fluorescence spectrum that is sensitive to redox status,wherein said mutant GFP shares at least 90% sequence identity with SEQ ID NO:
- 1 andwherein mutations at positions corresponding to SEQ ID NO;
1 include;
a) having a cysteine at one or both of the residues corresponding to 147 or 149 of SEQ ID NO;
1 andb) having a cysteine at one or both of the residues corresponding to 202 or 204 of SEQ ID NO;
1.
2 Assignments
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Accused Products
Abstract
The disclosure provides proteins that can be used to determine the redox status of an environment (such as the environment within a cell or subcellular compartment). These proteins are green fluorescent protein (GFP) variants (also referred to as redox sensitive GFP (rosGFP) mutants), which have been engineered to have two cysteine amino acids near the chromophore and within disulfide bonding distance of each other. Also provided are nucleic acid molecules that encode rosGFPs, vectors containing such encoding molecules, and cells transformed therewith. The disclosure further provides methods of using the rosGFPs (and encoding molecules) to analyze the redox status of an environment, such as a cell, or a subcellular compartment within a cell. In certain embodiments, both redox status and pH are analyzed concurrently.
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Citations
26 Claims
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1. An isolated mutant green fluorescent protein (GFP), with a fluorescence spectrum that is sensitive to redox status,
wherein said mutant GFP shares at least 90% sequence identity with SEQ ID NO: - 1 and
wherein mutations at positions corresponding to SEQ ID NO;
1 include;a) having a cysteine at one or both of the residues corresponding to 147 or 149 of SEQ ID NO;
1 andb) having a cysteine at one or both of the residues corresponding to 202 or 204 of SEQ ID NO;
1. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
- 1 and
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18. A mutant GFP, with a fluorescence spectrum that is sensitive to redox status,
wherein said mutant GFP shares at least 90% sequence identity with SEQ ID NO: - 1 and includes mutations selected from the group consisting of;
a) the residues corresponding to 147 and 204 of SEQ ID NO;
1 are cysteine and cysteine, respectively;b) the residues corresponding to 65, 147 and 204 of SEQ ID NO;
1 are threonine, cysteine, and cysteine respectively;c) the residues corresponding to 149 and 202 of SEQ ID NO;
1 are cysteine and cysteine, respectively;d) the residues corresponding to 65, 149 and 202 of SEQ ID NO;
1 are threonine, cysteine, and cysteine respectively;e) the residues corresponding to 147, 149, 202 and 204 of SEQ ID NO;
1 are cysteine, cysteine, cysteine and cysteine, respectively; andf) the residues corresponding to 65, 147, 149, 202 and 204 of SEQ ID NO;
1 are threonine, cysteine, cysteine, cysteine and cysteine, respectively.
- 1 and includes mutations selected from the group consisting of;
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19. A mutant GFP, with a fluorescence spectrum that is sensitive to redox status,
wherein said mutant GFP shares at least 95% sequence identity with SEQ ID NO: - 1 and includes mutations selected from the group consisting of;
a) the residues corresponding to 147 and 204 of SEQ ID NO;
1 are cysteine and cysteine, respectively;b) the residues corresponding to 65, 147 and 204 of SEQ ID NO;
1 are threonine, cysteine, and cysteine respectively;c) the residues corresponding to 149 and 202 of SEQ ID NO;
1 are cysteine and cysteine, respectively;d) the residues corresponding to 65, 149 and 202 of SEQ ID NO;
1 are threonine, cysteine, and cysteine respectively;e) the residues corresponding to 147, 149, 202 and 204 of SEQ ID NO;
1 are cysteine, cysteine, cysteine and cysteine, respectively; andf) the residues corresponding to 65, 147, 149, 202 and 204 of SEQ ID NO;
1 are threonine, cysteine, cysteine, cysteine and cysteine, respectively.
- 1 and includes mutations selected from the group consisting of;
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20. An isolated mutant GFP, with a fluorescence spectrum that is sensitive to redox status,
wherein said mutant GFP shares at least 95% sequence identity with SEQ ID NO: - 1 and
wherein mutations at positions corresponding to SEQ ID NO;
1 include;a) having a cysteine at one or both of the residues corresponding to 147 or 149 of SEQ ID NO;
1 andb) having a cysteine at one or both of the residues corresponding to 202 or 204 of SEQ ID NO;
1. - View Dependent Claims (21, 22, 23, 24, 25, 26)
- 1 and
Specification