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Methods for determining ligand binding to a target protein using a thermal shift assay

A target protein and protein technology, which is applied in the field of determining the binding of ligands to unpurified target proteins, can solve the problems of inability to use natural cells and tissues, affect the stability of GFP, and be unsatisfactory, and achieve the effect of easy multiplexing.

Active Publication Date: 2014-04-16
生物目标共享利益集团
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this approach is not ideal
First, the method requires the construction and expression of fusion proteins and therefore cannot be used in native cells and tissues but only in transformed cells
Furthermore, this method can only be used to detect ligand binding to proteins that are less stable than GFP
Finally, the use of additives or salts affects the stability of GFP, and therefore every experiment must use control GFP

Method used

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  • Methods for determining ligand binding to a target protein using a thermal shift assay
  • Methods for determining ligand binding to a target protein using a thermal shift assay
  • Methods for determining ligand binding to a target protein using a thermal shift assay

Examples

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Effect test

Embodiment 1- 4

[0119] The mensuration of embodiment 1-four kinds of test protein melting temperature

[0120] To determine the melting temperature of each protein in cells, three human soluble protein expression constructs in expression vectors with an N-terminal His-tag were used. This is done by exposing the protein-containing cells to a set of increasing temperatures, and after each temperature step, the cells are spotted onto a "lysis / filter sandwich" submerged in lysis buffer. By using this lysis / filtration step, soluble proteins (up to the protein's specific melting temperature) can be detected as dark spots on the capture nitrocellulose membrane, whereas precipitated proteins (i.e., above their specific melting temperature) cannot passed through the filter and was therefore not detected.

[0121] Materials and methods

[0122]In 96-well deep-well plates (Porvair Plc., UK), by inoculating 1 ml containing 50 μg / ml kanamycin (Sigma-Aldrich Co., USA) and 35 μg / ml chloride with frozen E....

Embodiment 2

[0125] Example 2 - Detection of Increased Melting Temperature After Ligand Binding

[0126] Post-addition and binding of either of two PIK3C3-specific inhibitors (wortmannin and compound 15e) to study PIK3C3 constructs with expression constructs of human soluble PIK3C3 protein in expression vectors with N-terminal His-tag Possible increase in melting temperature. After treatment with or without one of the two inhibitors, cells expressing the PIK3C3 construct were exposed to a set of increasing temperatures, and after each temperature step, the protein-expressing cells were spotted submerged in lysis buffer on the "lysing / filtering sandwich". By using this lysis / filtration step on a "lysis / filtration sandwich", soluble proteins (up to the specific melting temperature of the construct) can be detected as dark spots on capture nitrocellulose membranes, whereas precipitated proteins (i.e., high due to its specific melting temperature) does not pass through the filter and therefo...

Embodiment 3

[0131] Example 3 - Determination of the melting temperatures of four test proteins in mammalian cell systems

[0132] To determine the melting temperatures of the four proteins, lysates were prepared from incubated mammalian cells and exposed to a set of increasing temperatures. After the temperature step, the precipitated protein is removed, leaving only the soluble protein to be detected (i.e., up to the specific melting temperature of the protein).

[0133] Materials and methods

[0134]Lysates were prepared from incubated human adenocarcinoma cells (A549). Cells were disrupted on ice in hypotonic buffer and homogenized. The suspension was freeze-thawed multiple times and after complete lysis all insoluble aggregates and cell debris were pelleted by centrifugation. The supernatant containing the visually clear cytoplasmic protein fraction was aliquoted into 8-strip PCR tubes and subjected to a set of increasing temperatures. After heating for three minutes, the samples ...

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Abstract

The invention is directed to a method of determining whether a non-purified sample contains a target protein bound to a ligand of interest comprising the steps of: a) exposing said non-purified sample to a temperature which is capable of causing or enhancing precipitation of the unbound target protein to a greater extent than it is capable of causing or enhancing precipitation of the target protein bound to said ligand; b) processing the product of step a) in order to separate soluble from insoluble protein; and c) analysing either or both the soluble and insoluble protein fractions of step b) for the presence of target protein, wherein said target protein is not detected on the basis of enzymatic activity of a tag, peptide, polypeptide or protein fused thereto. Particularly, the invention may be used to determine whether drugs can bind to their protein targets in samples derived from patients to ascertain whether a certain drug can be used in a therapy for that patient. Additionally, the invention is directed to an instrument for use in the methods of the invention and use of a kit in the methods of the invention comprising an antibody and / or a non-protein fusion tag.

Description

technical field [0001] The present invention relates to methods for studying protein-ligand binding interactions, in particular by using thermal shift assays. [0002] More particularly, the invention relates to a method for assaying ligand binding to an unpurified target protein comprising the steps of heating the unpurified target protein and ligand and analyzing the product to detect soluble target protein. In certain embodiments, the methods of the invention use a separation step after heat treatment to separate soluble and insoluble proteins in order to estimate the amount of soluble target protein, and thus, heat stable ligand-binding target protein. The invention also relates to an apparatus for use in said method, comprising heating means, means for separating soluble and insoluble proteins and means for analyzing soluble and insoluble proteins for the presence of a target protein. Also described is the use of kits comprising antibodies or non-protein fusion markers i...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/9473G01N33/9446G01N33/946G01N33/9453G01N33/9486G01N33/68G01N33/9466G01N33/9493G01N33/948G01N33/6845C07K1/32G01N33/5375G01N33/6803
Inventor 帕尔·诺德隆德
Owner 生物目标共享利益集团
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