Zipper fastener structure for promoting formation of protein dimers and application thereof

A dimer and zipper technology, which is used in general structural and detailed detection of gas analyzers, expression-enhanced stability/folded protein fusion, antibody mimics/scaffolds, etc. out, inappropriate, etc.

Pending Publication Date: 2020-09-11
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 portion 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
[0012] CN102191209A discloses a VEGF165 and Ang-1 dual-gene co-expression vector and its application. In the dual-gene co-expression vector pAdTrack-CMV-Ang-1-IRES-VEFG165, human genes VEGF165 and Ang-1 enter through internal ribosomes The int...

Method used

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  • Zipper fastener structure for promoting formation of protein dimers and application thereof
  • Zipper fastener structure for promoting formation of protein dimers and application thereof
  • Zipper fastener structure for promoting formation of protein dimers and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0137] Construction of expression vector of zipper button dimer protein ESAT6-CFP10

[0138] 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:

[0139] The amino acid sequence of ESAT6 after expression is: DYKDDDDKGG-MAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGT AAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF-GGDDKDD;

[0140] The amino acid sequence of CFP10 after expression is: HHHHHHGG-MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAY QGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA-GGKKCKK;

[0141] The ESAT6 and CFP10 gene fragments after the sequence addition were synthesized and inserted into both ends of the IRES sequence in the pET expression vector to form an expression plasmid, and the purified expression plasmid was transformed into BL21(DE3) competent ce...

Embodiment 2

[0180] Example 2: Application of Cyclic Peptide Design in CCP Detection:

[0181] On the basis of the traditional CCP, the position of the disulfide bond is replaced, and after adding a dimeric zipper on both sides of the polypeptide, the new polypeptide structure is KKCK-CCP-DCDD; The detection rate of autoimmune antibodies has increased by 10%. It shows that the stability of the cyclic peptide 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.

[0182] Streptavidin-dimer zipper culinary peptide CCP was used for coating at a concentration of 5 μg / ml, and the samples were detected after blocking with skimmed milk powder. The secondary antibody was HRP-goat anti-human. The control reagent was the CCP ELISA detection kit from European Diagnostics.

[0183] like Figure 10 As shown, in 95 cases of RA patients, the detection rate o...

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Abstract

The invention belongs to the field of gene engineering, and relates to a zipper fastener structure for promoting formation of protein dimers and an application thereof, and the zipper fastener can beused for dimerization of homologous proteins and dimerization of heterologous proteins, and can also be used for polypeptide cyclization, dimerization of polypeptides and extension of polypeptides. Some examples can obtain an ESAT6-CFP10 dimer close to native conformation, wherein the dimer has better solubility and has a better stimulation effect on memory T cells than ESAT6-CFP10 protein expressed by linear fusion. The inventor also finds that the dimer zipper fastener can help to form more stable cyclic polypeptide, and the CCP polypeptide added with the dimer fastener can increase the detection rate of citrullinated antibody in serum of rheumatoid arthritis patients.

Description

technical field [0001] The invention relates to the field of biology, in particular to the field of proteins, in particular to a zipper button structure for promoting the formation of protein dimers and its application. Background technique [0002] A protein dimer is a protein quaternary structure. Homodimers are formed by combining two identical protein molecules (a process called homodimerization), while heterodimers are formed by two different proteins (called heterodimerization). dimerization). 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 specific regions that ensure the formation of dimerization (dimerization domains). In cell growth, reproduction, and signal transduction, protein dimerization plays an important role in t...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/35C07K14/00G01N33/68G01N33/569G01N33/543
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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