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Zipper fastener structure for promoting formation of protein dimer and application of zipper fastener structure

A technology of dimers and zipper buttons, which is applied in the detection of general structure details of gas analyzers, expression enhancement stability/folded protein fusion, antibody mimics/stents, etc., can solve problems such as excessive benefits and inappropriateness, and achieve The effect of reducing the chance of multimer formation, improving solubility, and improving purity

Pending Publication Date: 2022-07-29
GUANGZHOU LDEBIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fusion protein part of this structure is too large to be suitable as a dimeric fusion protein
Disulfide bonds are often used in the dimer design of proteins, but the problems they pose often outweigh the benefits

Method used

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  • Zipper fastener structure for promoting formation of protein dimer and application of zipper fastener structure
  • Zipper fastener structure for promoting formation of protein dimer and application of zipper fastener structure
  • Zipper fastener structure for promoting formation of protein dimer and application of zipper fastener structure

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Construction of expression vector of zipper-type dimer protein ESAT6-CFP10

[0104] A negatively charged amino acid group is added to the C-terminus of ESAT6 and a positively charged amino acid group is added to the C-terminus of the expression gene of CFP10, and a histidine purification tag is added to the front end of the CFP10, wherein:

[0105] The amino acid sequence of ESAT6 after expression is: DYKDDDDKGG (SEQ ID NO.: 3)-MAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF-GGDDCDD (SEQ ID NO.: 1);

[0106] The amino acid sequence of CFP10 after expression is: HHHHHHGG (SEQ ID NO.: 4) - MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA-GGKKCKK (SEQ ID NO.: 2);

[0107] The sequence-added ESAT6 and CFP10 gene fragments were synthesized and inserted into the two ends of the IRES sequence in the pET expression vector to form an expression plasmid. The purified expressio...

Embodiment 2

[0144] Example 2: Application of Cyclic Peptide Design in CCP Detection:

[0145] On the basis of the traditional CCP, the position of the disulfide bond was changed, and the dimer zipper was added on both sides of the polypeptide, and the new polypeptide structure was KKCK-CCP-DCDD; The detection rate of autoimmune antibodies in China has increased by 10%. It shows that the stability of cyclic formation is very important for the detection sensitivity of CCP, and increasing the stability of the cyclic peptide can further improve the sensitivity of CCP in the detection of citrullinated autoantibodies.

[0146] The samples were coated with streptavidin-dimer zipper loop peptide CCP at a concentration of 5µg / ml, and the samples were detected after blocking with non-fat milk powder. The secondary antibody was HRP-goat anti-human. The control reagent was the European Diagnostics CCP ELISA test kit.

[0147] like Figure 10 As shown, among 95 RA patients, the detection rate of CC...

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Abstract

The invention belongs to the field of genetic engineering, and relates to a zipper fastener structure for promoting formation of protein dimer and application thereof, and the zipper fastener structure can be used for dimerization of homoprotein and heteroprotein, and can also be used for polypeptide ring formation, dimerization polypeptide and polypeptide extension. Some examples can obtain the ESAT6-CFP10 dimer close to native conformation, the dimer has better solubility, and the memory T cell stimulation effect of the dimer is better than that of ESAT6-CFP10 protein of linear fusion expression. The inventor also finds that the dimer zipper buckle can help to form a more stable cyclic polypeptide, and the CCP polypeptide added with the dimer buckle can increase the detection rate of citrullinated autoantibody in the serum of a patient with rheumatoid arthritis.

Description

technical field [0001] The present invention relates to the biological field, in particular to the protein field, and in particular to a zipper button structure for promoting the formation of protein dimers and its application. Background technique [0002] Protein dimer is a protein quaternary structure. A homodimer is formed by the combination of two identical protein molecules (a process called homodimerization), while a heterodimer is formed by two different proteins (called a heterodimer) dimerization heterodimerization). Most dimers in biochemistry are not linked by covalent bonds. For example, reverse transcriptase is a non-covalently linked heterodimeric enzyme linked by two different amino acid chains. Another exception is the dimeric protein NEMO, a dimer linked by disulfide bonds. Some proteins contain special regions that ensure the formation of dimerization (dimerization regions). In cell growth, reproduction, and signal transduction, protein dimerization p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K14/00C07K14/35G01N33/543G01N33/569G01N33/68
CPCC07K14/35C07K14/00G01N33/6893G01N33/6863G01N33/6866G01N33/6869G01N33/5695G01N33/54306C07K2319/20C07K2319/21G01N2333/35G01N2333/47G01N2333/57G01N2333/5412G01N2333/5421G01N2333/525G01N2800/102C12N15/62G01N33/564C07K2319/73C07K2319/43C07K2319/35G01N2440/18C07K7/50G01N33/68
Inventor 杨翔楼建荣谢桂华
Owner GUANGZHOU LDEBIO TECH CO LTD
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