Known molecule and protein interaction detection system based on covalent linkage and identification or verification method thereof

A detection system and covalent connection technology, applied in the field of molecular biology, can solve the problems of complex operation and difficult weak interaction detection, and achieve the effect of simple operation, maintaining structure and activity, and accurate detection.

Active Publication Date: 2021-06-04
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The disadvantage is that it depends on the instrument, the operation is

Method used

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  • Known molecule and protein interaction detection system based on covalent linkage and identification or verification method thereof
  • Known molecule and protein interaction detection system based on covalent linkage and identification or verification method thereof
  • Known molecule and protein interaction detection system based on covalent linkage and identification or verification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Reactivity verification of PafA and Pup(E)

[0087] 1. Obtain GFP-Pup(E) and Pup(E)-GFP protein

[0088] Pup(E) refers to the mutation of glutamine (Q) at the C-terminus of the wild-type Pup molecule (sequence shown in SEQ NO.1) to glutamic acid (E). Pup(E) was fused and expressed at the N-terminus and C-terminus of the GFP protein respectively, and GFP-Pup(E) and Pup(E)-GFP were respectively constructed on pET28a and transformed into E.coli BL21(DE3) strain, wherein A 6×His tag was attached to the end without Pup(E). Culture 1L bacteria solution, OD 600 When ≈0.6, add IPTG, induce overnight at 18°C, and purify with nickel column to obtain GFP-Pup(E) and Pup(E)-GFP proteins.

[0089] 2. Acquisition of PafA enzyme

[0090] The wild-type PafA sequence is shown in SEQ NO.6. PafA was connected to pTrc99a vector and transformed into E.coliBL21(DE3) strain, wherein the C-terminus of PafA was connected with a 6×His tag. Culture 1L bacteria solution, OD 600 Whe...

Embodiment 2

[0093] Example 2: Transformation and Activity Verification of Streptavidin-Pup

[0094] 1. Obtain the modified streptavidin-Pup tetramer protein

[0095] In order to avoid streptavidin-Pup self-linkage (Pup sequence is shown in SEQ ID NO.1), the lysine on the surface of the streptavidin protein and the Pup molecule is mutated to arginine, and the mutated streptavidin Andin-Pup tetramer (SA m -Pup E ) amino acid sequence (SEQ ID NO.5) such as Figure 4 As shown in a (where Pup E The sequence is shown in SEQ ID NO.2). Will SA m -Pup E Constructed on pET28a vector and transformed into E.coli BL31(DE3) strain. Culture 1L bacteria solution, OD 600 When ≈0.6, add IPTG with a final concentration of 0.5mM, induce for 4 hours at 37°C, and use the inclusion body refolding method (Michael T.Jacobsen et al., 2017.Cell.Chem.Bio., 2017Aug 17; 24(8) :1040-1047) purified to obtain SA m -Pup E protein.

[0096] 2. Detection of SA m -Pup E biotin-binding activity, such as Figure...

Embodiment 3

[0101] Example 3: Transformation and Activity Verification of PafA Enzyme

[0102] 1. Obtain the transformed PafA enzyme, sequence (SEQ ID NO.7) such as Figure 5 as shown in a.

[0103] In order to avoid self-connection of PafA (sequence shown in SEQ ID NO.6), seven lysine sites on its surface were mutated to arginine, and the mutation sites were K162R, K202R, K320R, K361R, K423R, K435R and K446R. use Site-directed mutagenesis kit (Agilent) to construct 7 point-mutated PafA (named PafA 7KR ) was connected to pTrc99a vector and transformed into E.coli BL21(DE3) strain, wherein PafA 7KR A 6×His tag was attached to the C-terminus. Culture 1L bacteria solution, OD 600 When ≈0.6, add IPTG, induce overnight at 18°C, and purify with nickel column to obtain PafA 7KR enzyme.

[0104] 2. Detection of PafA 7KR Enzymes for their own Pupification activity, such as Figure 5 as shown in b.

[0105] will PafA 7KR Enzyme and SA m -Pup E or Pup E Co-incubated at 30°C for 4 ...

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Abstract

The invention discloses a known molecule and protein interaction detection system based on covalent linkage and an identification or verification method thereof. The detection system comprises: a) a streptavidin-oligopeptide tetramer; b) a PafA enzyme; and c) biotin-modified known molecules. Known molecules are labeled with biotin, after the known molecules interact with proteins, interaction proteins of the known molecules are efficiently captured through the streptavidin-oligopeptide tetramer under mild conditions, and then under catalysis of PafA enzyme, oligopeptide and the known molecules are in covalent linkage with the interaction proteins, therefore, non-covalent binding between the interacting known molecules and the protein is converted into covalent linkage between the streptavidin and the protein, and analysis, separation and identification are carried out. The method can realize capture of weak interaction and instantaneous interaction on the basis of keeping the natural structure of known molecules, and can be used for verification and discovery of known molecules and interaction proteins.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a detection system for the interaction between known molecules and proteins, in particular to a detection system for the interaction between known molecules and proteins based on covalent linkage and an identification or verification method thereof. Background technique [0002] Proteins are the executors of life activities. More than 80% of proteins function by interacting with other molecules, including a wide range of life processes such as embryonic development, cell communication, receptor-ligand binding, and signal transduction. Disordered, uncontrolled protein-molecule interactions may trigger cancer, neurodegenerative diseases, etc. (kesin et al., 2016. Chem. Rev., 116, 4884-4909). The interaction of small molecules or small molecule drugs with proteins in physiological processes has been widely studied in biomedicine and clinical applications, which will help to...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68G01N33/6845G01N33/6842G01N33/6848C12Q1/25G01N2333/9015G01N2440/36G01N33/58
Inventor 陶生策江何伟郑云萧陈红王雪宁
Owner SHANGHAI JIAO TONG UNIV
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