Internal protein tags

Patent metadata

Field	Value
Publication number	US-9969991-B2
Application number	US-201514852096-A
Country	US
Kind code	B2
Filing date	Sep 11, 2015
Priority date	Sep 12, 2014
Publication date	May 15, 2018
Grant date	May 15, 2018

Abstract

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.

First claim

We claim:

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.

Assignees

• Promega Corp

Inventors

• Dixon Andrew S
• Encell Lance P
• Machleidt Thomas
• Schwinn Marie
• Wood Keith
• Wood Monika
• Zimmerman Kris

Classifications

• C12Y308/01005

  Haloalkane dehalogenase (3.8.1.5) · CPC title

• C07K14/47

  from mammals · CPC title

• C12N9/14 Primary

  Hydrolases (3) · CPC title

• C07K14/00

  Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof · CPC title

• C07K2319/60

  containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] · CPC title