Nucleic acid encoding a self-assembling split-fluorescent protein system
View Patent ↗The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.
1. An isolated nucleic acid molecule comprising a polynucleotide sequence encoding beta strands 1-10 of a fluorescent protein comprising a polypeptide sequence at least 95% identical to:
the sequence set forth as SEQ ID NO: 4, wherein residue 39 is I, residue 105 is K, residue 111 is V, residue 128 is T, residue 166 is T, residue 167 is V and residue 205 is T; or the sequence set forth as SEQ ID NO: 6, wherein residue 80 is Q, residue 99 is Y, residue 105 is N, residue 111 is V, residue 128 is T, residue 166 is T, residue 172 is V and residue 205 is T; and
wherein the beta strands 1-10 of the fluorescent protein are capable of self-complementation with a polypeptide corresponding to a beta strand 11 of the fluorescent protein.
2. The nucleic acid molecule of claim 1 , wherein the beta strands 1-10 of the fluorescent protein comprise the amino acid sequence set forth as SEQ ID NO: 4.
3. The nucleic acid molecule of claim 1 , wherein the beta strands 1-10 of the fluorescent protein consist of the amino acid sequence set forth as SEQ ID NO: 4.
4. The nucleic acid molecule of claim 1 , wherein the beta strands 1-10 of the fluorescent protein comprise the amino acid sequence set forth as SEQ ID NO: 6.
5. The nucleic acid molecule of claim 1 , wherein the beta strands 1-10 of the fluorescent protein consist of the amino acid sequence set forth as SEQ ID NO: 4.
6. The nucleic acid molecule of claim 1 , wherein the polynucleotide sequence encoding beta strands 1-10 of the fluorescent protein comprises a nucleic acid sequence at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 3.
7. The nucleic acid molecule of claim 6 , wherein the polynucleotide sequence encoding beta strands 1-10 of the fluorescent protein comprises the nucleic acid sequence set forth as SEQ ID NO: 3.
8. The nucleic acid molecule of claim 6 , wherein the polynucleotide sequence encoding beta strands 1-10 of the fluorescent protein consists of the nucleic acid sequence set forth as SEQ ID NO: 3.
9. The nucleic acid molecule of claim 1 , wherein the polynucleotide sequence encoding beta strands 1-10 of the fluorescent protein comprises a nucleic acid sequence at least 95% identical to SEQ ID NO: 5.
10. The nucleic acid molecule of claim 9 , wherein the polynucleotide sequence encoding beta strands 1-10 of the fluorescent protein comprises the nucleic acid sequence set forth as SEQ ID NO: 5.
11. The nucleic acid molecule of claim 6 , wherein the polynucleotide sequence encoding beta strands 1-10 of the fluorescent protein consists of the nucleic acid sequence set forth as SEQ ID NO: 5.
12. The nucleic acid molecule of claim 1 , wherein the fluorescent protein is a Green Fluorescent Protein (GFP).
13. The nucleic acid molecule of claim 1 , operably linked to a promoter.
14. The nucleic acid molecule of claim 13 , wherein the promoter is an inducible promoter.
15. An expression vector comprising the nucleic acid molecule of claim 1 .
16. A host cell comprising the expression vector of claim 15 .
17. The host cell of claim 16 , wherein the host cell is an Escherichia coli cell.
18. The host cell of claim 16 , wherein the host cell is a eukaryotic cell.
19. The host cell of claim 16 , wherein the host cell is a mammalian cell.