DNA encoded compound screening method for identification tag-free protein or cell lysate with antibody

A technology of cell lysates and identification markers, which is applied in the field of antibody screening of DNA-encoded compounds targeting proteins without identification markers or cell lysates, which can solve the problems of proteins without post-translational modifications and cumbersome process of protein purification, etc., to improve screening efficiency , Avoid cumbersome processes and reduce screening costs

Active Publication Date: 2018-12-18
上海药明康德新药开发有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a DNA-encoded compound screening method for proteins without identification tags (tag) or cell lysates (lysates), which avoids the cumbersome process of purifying proteins and solves the problem that the purified proteins do not contain translation. The problem of post-modification can effectively improve the screening efficiency and reduce the screening cost

Method used

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  • DNA encoded compound screening method for identification tag-free protein or cell lysate with antibody
  • DNA encoded compound screening method for identification tag-free protein or cell lysate with antibody
  • DNA encoded compound screening method for identification tag-free protein or cell lysate with antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Screening of positive compounds on unmarked IDO1 to confirm the feasibility of the method

[0033] 1. Background: Indoleamine 2,3-dioxygenase IDO1 is the rate-limiting enzyme of tryptophan catabolism. In this case, the purchased unlabeled IDO1 was used as the target protein to verify the method.

[0034] 2. Implementation method:

[0035] Such as figure 1 As shown, the method specifically includes the following steps:

[0036] 1. Preparation of magnetic beads:

[0037] Vortex the Dynabeads magnetic beads for 30 seconds, pipette 20 μL into a 1.5ml Eppendorf tube (microcentrifuge tube, EP tube for short), place the EP tube on a magnetic stand to absorb the magnetic beads, and remove the supernatant. Wash the magnetic beads twice with 100 μL Antibody binding buffer (antibody binding buffer).

[0038] 2. Antibody binding:

[0039] Dilute 4 μg of IDO1 antibody with 100 μL Antibody binding buffer (antibody binding buffer), and incubate at room temperature for 30...

Embodiment 2

[0053] Example 2: DNA-encoded compound library screening for LAG3 expressed in cells

[0054] 1. Background: LAG3 is an immune checkpoint inhibitory receptor expressed on the surface of T cells. There are no small molecule compounds known to bind, nor are the binding proteins in the intracellular domain. Human LAG3 is a transmembrane protein, so it is difficult to express and purify it in bacteria or insect cells while retaining its physiological structure and activity. This method can isolate LAG3 and its bound protein from cells, and screen the DNA-encoded compound library for this complex.

[0055] 2. Implementation method:

[0056] Such as figure 1 As shown, the method specifically includes the following steps:

[0057] 1. Prepare cell lysate samples:

[0058] The human T cells expressing LAG3 were seeded into suspension cell culture flasks to make their growth saturated. Centrifuge at 800rpm for 10 minutes, discard the supernatant, wash the cells twice with 10ml DPB...

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Abstract

The invention discloses a DNA encoded compound screening method for an identification tag-free protein or cell lysate with an antibody. The method includes the steps of: (1) coating dynabead protein Gwith the antibody; (2) adding a solution containing the target protein into the antibody combined magnetic beads, and washing off the substances that do not combine with the antibody; (3) adding a DNA encoded compound library for screening; (4) cleaning and removing the uncombined compound; (5) extracting the uncombined compound by heating; and (6) decoding the compound structure by next-generation sequencing, and conducting synthesis and subsequent functional verification. The method provided by the invention avoids the tedious process of protein purification, at the same time solves the problem that the purified protein does not contain post-translational modification, and can effectively improve the screening efficiency and reduce the screening cost.

Description

technical field [0001] The invention belongs to the field of drug screening, and in particular relates to a method for screening DNA-encoded compounds, in particular to a method for screening DNA-encoded compounds using antibodies against unrecognized marker proteins or cell lysates. Background technique [0002] As a new type of small molecule drug screening method, DNA-encoded compound library can greatly improve the throughput and speed of drug screening, reduce manpower and material costs, and improve screening efficiency. The current method is commonly used to immobilize the purified target protein with an identification mark (tag) on ​​a solid phase carrier for compound screening, thus requiring the synthesis and purification process of the target protein, the process is cumbersome, time-consuming, high cost, and It is difficult for the purified protein to have post-translational modification under physiological conditions, so it is difficult to maintain the original p...

Claims

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Application Information

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IPC IPC(8): C40B30/04
CPCC40B30/04
Inventor 苏文姬李亚男杨传秀乐思远陆恒
Owner 上海药明康德新药开发有限公司
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