Internal protein tags
View Patent ↗Provided herein are substantially non-luminescent peptide/polypeptide tags that are inserted internally within a protein of interest or between N-terminal and C-terminal peptides/polypeptides. Interaction of the internally-inserted tag with a complement polypeptide/peptide that is also substantially non-luminescent results in the formation a bioluminescent reporter complex.
1. A polypeptide comprising:
an N-terminal segment of a protein of interest, a C-terminal segment of a protein of interest, and an internal tag between the N-terminal and C-terminal segments, wherein the internal tag comprises an amino acid sequence having less than 100% and greater than 45% sequence identity with SEQ ID NO: 2 inserted within a protein of interest; wherein a detectable bioluminescent signal is produced in the presence of a coelenterazine substrate when the internal tag contacts a polypeptide consisting of SEQ ID NO: 440 and wherein the polypeptide is not a naturally occurring polypeptide.
2. The polypeptide of claim 1 , wherein the N-terminal segment and the C-terminal segment are each at least 20 amino acids in length.
3. The polypeptide of claim 1 , wherein the amino acid sequence of the internal tag is selected from the peptides of Table 1.
4. The polypeptide of claim 1 , wherein the N-terminal segment and the C-terminal segment, if directly linked in the absence of the internal tag, comprise the sequence of a first protein of interest.
5. A nucleic acid comprising a sequence coding for a polypeptide of claim 1 .
6. A bioluminescent complex comprising:
(i) the polypeptide of claim 1 ; and
(ii) a second polypeptide comprising a complement polypeptide having less than 100% and greater than 45% sequence identity with SEQ ID NO: 440.
7. The bioluminescent complex of claim 6 , wherein the internal tag and the complement polypeptide have low affinity for each other.
8. The bioluminescent complex of claim 7 , wherein the second polypeptide is a fusion of the complement polypeptide and a second protein of interest.
9. The bioluminescent complex of claim 8 , wherein the fusion is an internal fusion or a traditional fusion.
10. The bioluminescent complex of claim 9 , wherein the second protein of interest has high affinity for all or a portion of the N-terminal segment and/or the C-terminal segment.
11. The bioluminescent complex of claim 7 , wherein the second polypeptide is linked to a molecule of interest.
12. The bioluminescent complex of claim 11 , wherein all or a portion of the N-terminal segment and/or the C-terminal segment has high affinity for the molecule of interest.
13. The bioluminescent complex of claim 6 , further comprising a coelenterazine substrate.
14. The bioluminescent complex of claim 6 , wherein the internal tag and the complement polypeptide have high affinity for each other.
15. The bioluminescent complex of claim 6 , wherein the second polypeptide is not a fusion polypeptide or linked to a molecule of interest.
16. The bioluminescent complex of claim 6 , wherein the amino acid sequence of the complement polypeptide is selected from the polypeptides of Table 2.