Protein/polypeptide sequencing method adopting Aerolysin nanopores

A nano-pore, protein technology, applied in the biological field, can solve the problems of inability to effectively identify 20 amino acids, difficulty in obtaining amino acid sequence information, lack of efficient organic fluorophores, etc., and achieve the effect of improving amino acid identification ability.

Pending Publication Date: 2021-03-12
NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing fluorescent sequencing methods lack efficient organic fluorophores for the detection of 20 different amino acids without significant overlap between emission peaks to specifically label the 20 amino acids
However, the sub-nanometer-scale tunneling measurement interface used in the tunneling current detection technology is difficult to stably prepare. These challenges make it impossible to effectively identify 20 amino acids and their post-translational modifications, although the existing technology can distinguish several amino acids. , it is more difficult to obtain amino acid sequence information
Therefore, single-molecule protein sequencing is still facing huge challenges. It is urgent to develop new principles for sensitive detection of 20 amino acid sequence information, and to establish innovative methods for precise measurement of amino acid sequence and post-translational modifications of a single protein molecule.

Method used

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  • Protein/polypeptide sequencing method adopting Aerolysin nanopores
  • Protein/polypeptide sequencing method adopting Aerolysin nanopores
  • Protein/polypeptide sequencing method adopting Aerolysin nanopores

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Embodiment 1

[0065] The invention discloses a method for sequencing polypeptide molecules of a cysteine-specific Aerolysin nanopore. The polypeptide uses Glu as a guide chain, and the amino acid sequences of the two polypeptide molecules are Glu-Gly-Cys and Glu-Cys-Gly respectively. Specific steps are as follows:

[0066] (1) Two electrical primary screening channels, N226Q and T232K, were designed, and the corresponding mutant Proerolysin protein was expressed and purified by site-directed mutagenesis technology for channel construction. For specific steps, refer to patent CN202010131704.8.

[0067] (2) Mix 1 mg / mL Proerolysin protein with trypsin 10:1 and incubate at room temperature for 6 hours to obtain Aerolysin monomer protein with pore-forming activity.

[0068] (3) Control the experimental temperature at 22±1°C. Add 1mL (1.0 M KCl, 10 mMTris, 1.0 mM EDTA, pH=8) buffer solution into the two detection cells respectively, and prepare the phospholipid bilayer by pulling method. For sp...

Embodiment 2

[0074] A method for detecting phosphorylated polypeptides using mutant Aerolysin nanopores, using S-K-I-G as the guide strand, the template polypeptide sequence is S-K-I-G-S-T-E-N-L, and phosphorylated modified sequences at the fifth serine and the sixth threonine respectively S-K-I-G- p S-T-E-N-L and S-K-I-G-S- p T-E-N-L. Specific steps are as follows:

[0075] (1) The wild-type electrical primary screening channel was designed, and the wild-type Proerolysin protein was expressed and purified for channel construction. For specific steps, refer to patent CN202010131704.8.

[0076] (2) Mix 1 mg / mL Proerolysin protein with trypsin 10:1 and incubate at room temperature for 6 hours to obtain Aerolysin monomer protein with pore-forming activity.

[0077] (3) Control the experimental temperature at 22±1°C. Add 1mL (1.0 M KCl, 10 mMTris, 1.0 mM EDTA, pH=8) buffer solution into the two detection cells respectively, and prepare the phospholipid bilayer by pulling method. For specif...

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Abstract

The invention provides a method for protein/polypeptide sequencing based on Aerolysin nanopores. The method realizes specific resolution of natural amino acid and post-translational modification thereof and accurate acquisition of a single-molecule protein sequence, and comprises the following steps: (1) carrying out protein unfolding; (2) marking an end position sequencing starting point; (3) carrying out electrified preliminary screening; (4) unfolding a polypeptide tertiary structure; (5) carrying out amino acid orthogonal recognition; (6) carrying out limited range perturbation assisted amino acid recognition; and (7) carrying out single molecule protein sequence determination. According to the method, an innovative method for accurate measurement of single protein molecule amino acidsequence and post-translational modification is established for sensitive detection of information of 20 amino acid sequences.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a protein / polypeptide sequencing method based on Aerolysin nanopores. Background technique [0002] Thousands of different proteins maintain all the functions of cells. The precise determination of the amino acid sequences of proteins in organisms can provide a basis for understanding protein functions, the biological processes they participate in, and the interactions between proteins and proteins (or other biomolecules). Key Information. According to the transmission direction of genetic information, protein synthesis is synthesized by processes such as DNA transcription, post-transcriptional processing, translation, and post-translational modification. However, a gene can be spliced ​​in multiple mRNA forms during transcription, and the same protein can undergo post-translational modification in many forms, so there is a large difference between the genotype and pheno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/113C07K1/12
CPCC07K1/1133C07K1/128G01N33/48721C07K1/12
Inventor 龙亿涛
Owner NANJING UNIV
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