Highly thermostable fluorescent proteins
View Patent ↗Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99° C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80° C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.
1. A nucleic acid molecule comprising a polynucleotide encoding the polypeptide having the amino acid sequence of SEQ ID NO: 9.
2. The nucleic acid molecule of claim 1 , wherein the amino acid sequence of SEQ ID NO: 9 is encoded by the polynucleotide of SEQ ID NO: 4.
3. The nucleic acid molecule of claim 2 , which is a vector.
4. The nucleic acid molecule of claim 2 , which is an expression vector.
5. A host cell comprising the nucleic acid molecule of claim 1 .
6. A host cell comprising the vector of claim 3 .
7. A host cell comprising the expression vector of claim 4 .
8. A method of making a polypeptide having the amino acid sequence of SEQ ID NO: 9, the method comprising expressing the nucleic acid molecule of claim 1 .
9. The method of claim 8 , wherein the nucleic acid molecule is expressed in a host cell.
10. The method of claim 8 , wherein the nucleic acid molecule comprises the sequence of SEQ ID NO: 4.
11. The method of claim 8 , wherein the nucleic acid molecule is a vector.
12. The method of claim 8 , further comprising isolating the expressed polypeptide.
13. A method of making a fluorescent polypeptide, comprising growing the host cell of claim 7 under conditions sufficient to express the polypeptide with the amino acid sequence of SEQ ID NO: 9.