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Recombinant protein and application thereof for detecting trimethylation modification of histone sites

A technology of recombinant protein and histone, which is applied in the field of protein modification detection, can solve the problems of high price of commercial antibodies, long antibody preparation cycle, unstable batch quality, etc., to improve the level of soluble expression, facilitate purification and detection, low cost effect

Active Publication Date: 2019-12-20
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is that the commercialized antibodies targeting histone trimethylation modification sites are expensive, the quality of each batch is unstable, the cross-reaction is obvious, the preparation cycle of antibodies modified at specific sites is long, and the yield is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Preparation of recombinant protein GST-Tud01

[0066] 1) The whole gene was optimized to synthesize the nucleotide sequence described in SEQ ID NO.1, that is, the nucleotide sequence of Tud01, and restriction enzyme sites BamHI and XhoI were introduced at both ends of the gene, and the synthesized gene was cloned into pUC57 in the carrier;

[0067] 2) Extract the vector containing the Tud01 sequence in step 1), double enzyme digestion, connect to the same double enzyme digestion pGEX-4t-1 vector, and obtain the recombinant vector pGEX-4t-1-Tudo01 after screening and identification; it can encode SEQ The amino acid sequence described in ID No.2;

[0068] 3) Transform Escherichia coli competent cells BL21 with the recombinant vector pGEX-4t-1-Tud01, and activate the monoclonal into 5ml LB liquid medium. When OD600=0.6, add 0.4mM IPTG, induce 12-15h at 16-20 degrees, and you can get Soluble expression of the recombinant protein.

[0069] 4) Obtain about 15-20 mg of GST-...

Embodiment 2

[0071] Preparation of recombinant protein GST-Tud02

[0072] 1) The whole gene was optimized to synthesize the nucleotide sequence described in SEQ ID NO.3, which is the nucleotide sequence of Tud02, and restriction enzyme sites BamHI and XhoI were introduced at both ends of the gene, and the synthesized gene was cloned into pUC57 in the carrier;

[0073] 2) Extract the vector containing the Tud02 sequence in step 1), double enzyme digestion, and connect to the same double enzyme digestion pGEX-4t-1 vector, after screening and identification, the recombinant vector pGEX-4t-1-Tud02 can encode SEQ ID The amino acid sequence described in No.4;

[0074] 3) Transform Escherichia coli competent cells BL21 with the recombinant vector pGEX-4t-1-Tud01, activate the monoclonal into 5ml LB liquid medium, add 0.4mM IPTG when OD600=0.6, and induce at a low temperature of 16°C to 20°C for 12 to 15h, Soluble expression of the recombinant protein can be obtained;

[0075] 4) Obtain about 1...

Embodiment 3

[0077] Sensitive detection of recombinant proteins

[0078] ITC equipment: The titration was performed using MicroCal iTC200system(GE Healthcare)

[0079] For the experimental method, refer to the instrument instructions and standard settings.

[0080] Main steps: determine appropriate reactant concentrations, prepare samples; titrate, collect calorific data; calibrate data, fit regression, calculate thermodynamic parameters, and finally analyze the model.

[0081] 1) All test samples were placed at a constant temperature of 25°C for reaction;

[0082] 2) Synthesize 10 mg of the polypeptide to be tested (trimethylation modification on the 4th lysine of histone H3 and the 20th lysine of histone H4), and dissolve it with ITC basic buffer. The mother liquor concentration is 0.5mg / ml;

[0083] Peptide sequence 1 is ARTKme3QTARKS

[0084] In the ITC binding experiment, the recombinant proteins corresponding to SEQ ID No.2 and 4 (i.e. the recombinant proteins in Examples 1 and 2...

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Abstract

The invention provides a recombinant protein for detecting tri-methylated modification of a histone locus and application thereof. An amino acid sequence of the recombinant protein comprises an N-terminal GST fusion protein sequence and a C-terminal Tudor structural domain sequence which are connected in series. The recombinant protein provided by the invention has high affinity to H3K4 and H4K20, can be used for replacing a commercialized antibody to perform Western Blot experiment, is high in sensitivity, has no cross linking; the production cost is remarkably reduced, the batch quality is extremely stable, and the batch production can be realized quickly.

Description

technical field [0001] The invention relates to the technical field of protein modification detection, and more specifically, to a recombinant protein for detecting trimethylation modification of histone sites and its application. Background technique [0002] Histones are an important building block of chromosomes. Histones undergo more than 100 different post-translational modifications, such as phosphorylation, acetylation, methylation, ubiquitination, and glycosylation. These post-translational modifications are an important way to regulate the structure and function of chromosomes, and play a key role in regulating many physiological processes such as development, metabolism, and disease. It is of great significance to study these histone modifications, and the systematic positioning of these histone modifications has become the focus of many major international projects. Genome-wide localization of these histone modifications requires specific antibodies against thes...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K14/00C12N15/70G01N33/68C07K16/00C12R1/19
CPCC07K14/00C07K16/00C07K2319/23C12N15/70G01N33/68
Inventor 李珊珊
Owner HUBEI UNIV
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