Activation of bioluminescence by structural complementation

Patent metadata

Field	Value
Publication number	US-9797890-B2
Application number	US-201414209610-A
Country	US
Kind code	B2
Filing date	Mar 13, 2014
Priority date	Mar 15, 2013
Publication date	Oct 24, 2017
Grant date	Oct 24, 2017

Abstract

Provided herein are compositions and methods for the assembly of a bioluminescent complex from two or more non-luminescent (e.g., substantially non-luminescent) peptide and/or polypeptide units. In particular, bioluminescent activity is conferred upon a non-luminescent polypeptide via structural complementation with another, complementary non-luminescent peptide.

First claim

The invention claimed is:

1. A peptide comprising an amino acid sequence having greater than 45% but not greater than 90% sequence identity with SEQ ID NO: 2, wherein a bioluminescent signal produced in the presence of a furimazine substrate is substantially increased when the peptide contacts a polypeptide consisting of SEQ ID NO: 440 when compared to a bioluminescent signal produced by the peptide and furimazine substrate alone, and wherein the amino acid sequence is not a naturally occurring protein or a fragment thereof.

2. The peptide of claim 1 , wherein the bioluminescent signal is substantially increased when the peptide associates with the polypeptide consisting of SEQ ID NO: 440.

3. The peptide of claim 1 , wherein the peptide exhibits enhancement of one or more traits compared to a peptide of SEQ ID NO: 2, wherein the traits are selected from: affinity for the polypeptide consisting of SEQ ID NO: 440, expression, intracellular solubility, intracellular stability, and bioluminescent activity when combined with the polypeptide consisting of SEQ ID NO: 440.

4. The peptide of claim 1 , wherein the amino acid sequence is selected from the peptides of Table 1.

5. The peptide of claim 1 , wherein the amino acid sequence is synthetic, contains non-natural amino acids or is a peptide mimic.

6. A fusion polypeptide comprising the peptide of claim 1 and a first interaction polypeptide that is configured to form a complex with a second interaction polypeptide upon contact of the first interaction polypeptide and the second interaction polypeptide.

7. A bioluminescent complex comprising:
(a) the fusion polypeptide of claim 6 ; and
(b) a second fusion polypeptide comprising:
(i) the second interaction polypeptide, and
(ii) a complement polypeptide that emits a detectable bioluminescent signal in the presence of a substrate when associated with a peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2;
wherein the fusion polypeptide of claim 6 and the second fusion polypeptide are associated;
and wherein the peptide comprising an amino acid sequence having greater than 45% but not greater than 90% sequence identity with SEQ ID NO: 2 and the complement polypeptide are associated.

8. A bioluminescent complex comprising a non-covalent association of:
(a) a peptide comprising a peptide amino acid sequence having greater than 45% but not greater than 90% sequence identity with SEQ ID NO: 2, wherein the peptide amino acid sequence is not a naturally occurring protein or a fragment thereof; and
(b) a polypeptide comprising a polypeptide amino acid sequence having greater than 40% but not greater than 95% sequence identity with SEQ ID NO: 440, wherein the polypeptide amino acid sequence is not a naturally occurring protein or a fragment thereof, and wherein the bioluminescent complex exhibits detectable luminescence;
wherein the bioluminescent complex produces substantially increased luminescence in the presence of a furimazine substrate when compared to either the peptide or polypeptide alone in the presence of the furimazine substrate.

9. The bioluminescent complex of claim 8 , wherein the peptide amino acid sequence is selected from the peptide sequences of Table 1.

10. The bioluminescent complex of claim 8 , wherein the polypeptide amino acid sequence is selected from the polypeptide sequences of Table 2.

11. The bioluminescent complex of claim 8 , wherein the peptide amino acid sequence is attached to a first interaction element that is associated with a second interaction element.

12. The bioluminescent complex of claim 11 , wherein the polypeptide amino acid sequence is attached to the second interaction element.

13. The bioluminescent complex of claim 12 , wherein the polypeptide amino acid sequence and peptide amino acid sequence are substantially non-luminescent in the absence of the association of the first and second interaction elements.

Assignees

• Promega Corp

Inventors

• Dixon Andrew S
• Encell Lance
• Hall Mary
• Wood Keith
• Wood Monika
• Schwinn Marie
• Binkowski Brock F
• Zegzouti Hicham
• Nath Nidhi
• Mondal Subhanjan
• Goueli Said
• Meisenheimer Poncho
• Kirkland Thomas
• Unch James
• Pulukkunat Dileep K
• Robers Matthew
• Dart Melanie
• Machleidt Thomas

Classifications

• G01N33/581

  with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates) · CPC title

• C12Q1/66

  involving luciferase · CPC title

• G01N33/542

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title

• C07K14/43509

  from crustaceans · CPC title

• G01N33/533 Primary

  with fluorescent label · CPC title