Methods for dye selection for protein melt temperature determinations

Patent metadata

Field	Value
Publication number	US-10866245-B2
Application number	US-201816001613-A
Country	US
Kind code	B2
Filing date	Jun 6, 2018
Priority date	Mar 15, 2013
Publication date	Dec 15, 2020
Grant date	Dec 15, 2020

Abstract

According to the present teachings, compositions, kits, and methods for protein melt analysis are provided that utilizing one or more fluorophore dyes. In some embodiments, a method comprises preparing a sample by mixing at least one protein with two or more dyes, and applying a controlled heating, while recording the fluorescence emission of the sample. The methods can be used, for example, for screening conditions for optimized protein stability, screening for ligands that bind and enhance protein stability (e.g., protein-protein interactions), screening for mutations for enhanced stability, screening crystallization conditions for protein stability, screening storage conditions for protein stability, and screening conditions in which a protein will be used (e.g., production conditions, treatment conditions, etc.) for protein stability.

First claim

What is claimed is:

1. A method of measuring the stability of at least one protein, comprising:
(a) forming a sample solution mixture comprising at least one protein and a mixture comprising at least two fluorophore dyes, wherein one of the at least two fluorophore dyes is selected from the group consisting of SYPRO Orange, SYPRO Red, and SYPRO Tangerine, and the other one of the at least two fluorophore dyes is selected from the group consisting of SYPRO Orange, SYPRO Red, SYPRO Tangerine, and Nile Red;
wherein each of the at least two fluorophore dyes is selected such that they are spectrally distinct;
(b) applying a controlled heating to the mixture; and
(c) measuring fluorescence emitted over a temperature range.

2. The method of claim 1 , wherein the sample solution mixture is formed when the at least one protein is in its native state.

3. The method of claim 1 , wherein when the at least one protein is present in its native state, then each of the at least two fluorophore dyes is configured to provide at least a minimally fluorescent signal and when the at least one protein is present in an unfolded state, then each of the at least two fluorophore dyes is configured to provide a substantially increased fluorescent signal.

4. The method of claim 1 , wherein the measuring step is performed in the presence of a filter.

5. The method of claim 1 , wherein the other one of the at least two fluorophore dyes is selected from Nile Red.

6. The method of claim 1 , further comprising the step of (d) calculating a T m of the at least one protein using the measured fluorescence.

7. The method of claim 1 , wherein the sample solution mixture further comprises a ligand which is configured to form a protein/ligand complex with the at least one protein.

8. The method of claim 7 , wherein the method further comprises the steps of:
(e) performing the steps (a)-(c) wherein the sample solution mixture contains only the at least one protein;
(f) calculating a T m of the at least one protein using the measured fluorescence;
(g) performing the steps (a)-(c) wherein the sample solution mixture contains the at least one protein and a ligand in a protein/ligand complex;
(h) calculating a T m of the at least one protein and the ligand using the measured fluorescence of step (g); and
(i) comparing the T m obtained in step (f) and the T m obtained in step (h); and
(j) thereby analyzing the change in stability upon forming the protein/ligand complex with the at least one protein.

9. The method of claim 1 , wherein the controlled heating is a thermal ramp.

Assignees

• Life Technologies Corp

Inventors

• Bornarth Carole
• Rath Mousumi

Classifications

• G01N33/582 Primary

  with fluorescent label · CPC title

• C09B23/107

  four >CH- groups · CPC title

• C09B23/10

  The polymethine chain containing an even number of >CH- groups {(styryl dyes C09B23/14, C09B23/14 takes precedence)} · CPC title

• G01N33/6803

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• C09B23/145

  the ethylene chain carrying an heterocyclic residue, e.g. heterocycle-CH=CH-C6H5 · CPC title