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Method using implicit peptides trans splicing to manufacture protein molecular weight standard

A molecular weight standard, protein technology, applied in the field of biological science and biology, can solve the problem of high production cost

Inactive Publication Date: 2008-10-08
NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Some large biological companies obtain integer size molecular weight standards by directly expressing specific recombinant proteins, but this method leads to extremely high production costs, such as Invitrogen's BenchMark Protein Ladder, the price is 2600 yuan per ml

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  • Method using implicit peptides trans splicing to manufacture protein molecular weight standard
  • Method using implicit peptides trans splicing to manufacture protein molecular weight standard
  • Method using implicit peptides trans splicing to manufacture protein molecular weight standard

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Embodiment Construction

[0018] For example, DnaE and DnaB of the cyanobacteria Synechocystis sp. PCC6803 decompose intein A and B into amino terminal (N terminal) and carboxyl terminal (C terminal), namely An, Ac, Bn, Bc. They are then combined into five recombinant proteins, starting point (X-An), middle segment 1 (Ac-X-Bn), middle segment 2 (Bc-X-An), end point 1 (Ac-X), end point 2 (Bc- X).

[0019] First let the "starting point" protein react with the "end point 2" protein to obtain a protein with twice the molecular weight of the known protein;

[0020] If the "starting point" protein and the "middle segment 2" protein are reacted, and then the "end point 1" protein is added to the reaction, a protein with a molecular weight three times that of the known protein can be obtained;

[0021] If the "starting point" protein and the "middle segment 1" protein are reacted, then the "middle segment 2" protein is added for reaction, and the "end point 2" protein is finally added, a protein with a molecu...

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Abstract

The invention discloses a method utilizing internal peptide inverse splicing to prepare protein molecular weight standard, which includes: selecting a section of known protein X with a certain molecular weight, selecting two internal peptides A and B not compatible to each other, and then respectively decompounding internal peptides A and B into amino terminal (N terminal) and carboxyl terminal (C terminal), namely An, Ac, Bn, Bc; and then using them to consist five recombinant proteins, namely an initial point protein (X-An), an intermediate section 1 protein(Ac-X-Bn), an intermediate section 2 protein(Bc-X-An), an end point 1 protein(Ac-X), and an end point 2 protein(Bc-X); obtaining protein molecular weight standard with multiple molecular weight distribution by mutually combining, connecting and overlapping the five proteins.

Description

technical field [0001] The invention relates to a method for preparing square protein molecular weight standards, which can be used in the fields of biological sciences, biological methods and the like. Background technique [0002] 1. Intein-mediated trans-splicing principle [0003] Intein (intein) is a polypeptide chain parasitic in other proteins. Its coding DNA sequence is transcribed and translated together with the exons of the normal protein to produce a polypeptide chain. Then cut out the sequence corresponding to the intein, and then connect the sequences of the parasitized protein to become a functional protein. This is a protein self-splicing mechanism. Generally speaking, intein has 10 motifs (motif), the order is N□AN 2 BN 4 CDEHFG C□, where A, N 2 , B, N 4 , F, and G belong to the motif of the self-splicing domain, while C, D, E, and H belong to the motif of the homing endonuclease domain (Perler FB, Olsen GJ, AdamE. Compilation and analysis of intein s...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N1/28
Inventor 王强陈建群张云华张鹏飞王凯刘静王海燕李宕
Owner NANJING UNIV
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