Activation of bioluminescence by structural complementation

Patent metadata

Field	Value
Publication number	US-10648971-B2
Application number	US-201816035117-A
Country	US
Kind code	B2
Filing date	Jul 13, 2018
Priority date	Mar 15, 2013
Publication date	May 12, 2020
Grant date	May 12, 2020

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 70% sequence identity with SEQ ID NO: 390 or SEQ ID NO: 2271, but less than 100% sequence identify with SEQ ID NO: 2, wherein a bioluminescent signal produced in the presence of a furimazine substrate is 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 of the peptide 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 synthetic, contains non-natural amino acids or is a peptide mimic.

5. A bioluminescent complex comprising:
(a) the peptide of claim 1 ; and
(b) a polypeptide comprising an amino acid sequence having greater than 40% but less than 100% sequence identity with SEQ ID NO: 440;
wherein a bioluminescent signal produced by the bioluminescent complex in the presence of a furimazine substrate is substantially increased when compared to a bioluminescent signal produced in the presence of furimazine substrate by the peptide or the polypeptide alone.

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 a polypeptide comprising an amino acid sequence having greater than 40% but less than 100% sequence identity with SEQ ID NO: 440;

wherein a bioluminescent signal produced by the bioluminescent complex in the presence of a furimazine substrate is substantially increased when compared to a bioluminescent signal produced in the presence of furimazine substrate by the peptide or the polypeptide alone.

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

• G01N33/542

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

• G01N33/581

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

• C07K7/08 Primary

  having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68) · CPC title

• G01N33/533 Primary

  with fluorescent label · CPC title

• C07K14/43509

  from crustaceans · CPC title