Circular permutant GFP insertion folding reporters
Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.
1. An isolated nucleic acid, comprising:
a polynucleotide encoding a fluorescent protein circular permutant derived from superfolder Green Fluorescent Protein (GFP); and
an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, which when expressed is capable of reporting on the degree to which a polypeptide encoded by the inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.
2. The nucleic acid of claim 1 , wherein the cloning site is located between the coding sequences defining the native N- and C-termini of the fluorescent protein.
3. The nucleic acid of claim 2 , wherein the cloning site is located within a polynucleotide encoding a conformationally flexible polypeptide linker oriented between the native N- and C-termini of the fluorescent protein.
4. The nucleic acid of claim 3 , wherein the linker comprises the amino acid sequence of SEQ ID NO: 25.
5. The nucleic acid of claim 3 , wherein the cloning site is flanked by two linkers comprising the amino acid sequence of SEQ ID NO: 25.
6. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6.
7. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding amino acid residues 13-274 of SEQ ID NO: 6.
8. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8.
9. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding amino acid residues 13-274 of SEQ ID NO: 8.
10. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 10.
11. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding amino acid residues 13-274 of SEQ ID NO: 10.
12. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14.
13. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding amino acid residues 13-274 of SEQ ID NO: 14.
14. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16.
15. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding amino acid residues 13-274 of SEQ ID NO: 16.
16. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 18.
17. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding amino acid residues 13-274 of SEQ ID NO: 18.
18. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 20.
19. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding amino acid residues 13-274 of SEQ ID NO: 20.
20. The nucleic acid of claim 1 , comprising the nucleotide sequence of SEQ ID NO: 5.
21. The nucleic acid of claim 1 , comprising nucleotide residues 37-822 of SEQ ID NO: 5.
22. The nucleic acid of claim 1 , comprising the nucleotide sequence of SEQ ID NO: 7.
23. The nucleic acid of claim 1 , comprising nucleotide residues 37-822 of SEQ ID NO: 7.
24. The nucleic acid of claim 1 , comprising the nucleotide sequence of SEQ ID NO: 9.
25. The nucleic acid of claim 1 , comprising nucleotide residues 37-822 of SEQ ID NO: 9.
26. The nucleic acid of claim 1 , comprising the nucleotide sequence of SEQ ID NO: 11.
27. The nucleic acid of claim 1 , comprising nucleotide residues 37-822 of SEQ ID NO: 11.
28. The nucleic acid of claim 1 , comprising the nucleotide sequence of SEQ ID NO: 13.
29. The nucleic acid of claim 1 , comprising nucleotide residues 37-822 of SEQ ID NO: 13.
30. The nucleic acid of claim 1 , comprising the nucleotide sequence of SEQ ID NO: 15.
31. The nucleic acid of claim 1 , comprising nucleotide residues 37-822 of SEQ ID NO: 15.
32. The nucleic acid of claim 1 , comprising the nucleotide sequence of SEQ ID NO: 17.
33. The nucleic acid of claim 1 , comprising nucleotide residues 37-822 of SEQ ID NO: 17.
34. The nucleic acid of claim 1 , comprising the nucleotide sequence of SEQ ID NO: 19.
35. The nucleic acid of claim 1 , comprising nucleotide residues 37-822 of SEQ ID NO: 19.
36. An expression vector comprising a nucleic acid according to claim 1 , which is capable of expressing the fluorescent protein circular permutant encoded therein in a host cell.
37. The vector of claim 36 , which is a pET vector.
38. The vector of claim 37 , wherein the pET vector has the nucleotide sequence of SEQ ID NO: 21, and wherein a nucleic acid according to any one of claim 6 , 8 , 10 , 12 , 14 , 16 , 18 , 20 , 22 , 24 , 26 , 28 , 30 , 32 or 34 is inserted between the NcoI and KpnI restriction sites therein.
39. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 12.
40. The nucleic acid of claim 1 , comprising a polynucleotide sequence encoding amino acid residues 13-274 of SEQ ID NO: 12.