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Recombinant protein used for detecting acetylation of histone loci and application of recombinant protein

A technology of recombinant protein and histone, which is applied in the field of protein modification detection, can solve the problems of long antibody preparation cycle, unstable batch quality, serious cross-reaction, etc., and achieve mass production, low experimental detection cost, and homogeneous good sex effect

Active Publication Date: 2017-10-24
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is that the commercialized antibodies targeting histone acetylation modification sites are expensive, the quality of each batch is unstable, the cross-reaction is serious, the preparation cycle of specific site-modified antibodies is long, and the yield is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Preparation of recombinant protein GST-BBM2

[0054] 1) Firstly, the nucleotide sequence described in SEQ ID No.1 sequence is optimized and synthesized from the whole gene, and the translated amino acid sequence is the double-domain BBM2. Restriction sites BamHI and XhoI were introduced at both ends of the gene, and the synthesized gene was cloned into the pUC57 vector;

[0055] 2) Extracting the vector containing the BBM2 sequence in step 1), double digestion, connecting to the same double digestion pGEX-4t-1 vector, screening and identifying the recombinant vector, named pGEX-4t-1-BBM2;

[0056] 3) Transform Escherichia coli competent cells BL21 with the recombinant vector pGEX-4t-1-BBM2, and activate the monoclonal into 5ml LB liquid medium. When OD600=0.6, add 0.5mM IPTG, and induce 12-15 cells at a low temperature of 16°C-20°C hours, the soluble expression of the recombinant protein can be obtained;

[0057] 4) Obtain about GST-BBM3 recombinant protein from 1 L o...

Embodiment 2

[0059] Specific detection of recombinant proteins

[0060] ITC equipment: The titration was performed using MicroCal iTC200 system (GE Healthcare)

[0061] For the experimental method, refer to the instrument instructions and standard settings.

[0062] Main steps: determine appropriate reactant concentrations, prepare samples; titrate, collect calorific data; calibrate data, fit regression, calculate thermodynamic parameters, and finally analyze the model.

[0063] 1) All test samples were placed at a constant temperature of 25°C for reaction;

[0064] 2) Synthesize 10 mg of the polypeptide to be tested (modified by acetylation on the 14th lysine of histone H3), and dissolve it with ITC basic buffer. The concentration of the mother liquor is 0.5mg / ml.

[0065] The peptide sequence is derived from H3K14ac, which is ARKSTGGKacAPRKQ

[0066] Dilute solution: 20mM Tris-HCl, 50mM NaCl, pH7.5, peptide working concentration 0.8-1.2mM;

[0067] 3) The recombinant protein was mea...

Embodiment 3

[0073] Detected with recombinant protein GST-BBM2 protein immunoblotting WB (Western Blot) described in SEQ ID No.2

[0074] 1) Use RIPA lysate to extract human liver tissue nuclear protein. Use 15% SDS-PAGE polyacrylamide gel electrophoresis to separate the protein in the sample, and then electrotransfer the protein to the nitrocellulose membrane;

[0075] 2) Block with 5% BSA or skimmed milk powder at 37 degrees for 1 to 2 hours, wash 3 times with TBST, 5 minutes each time; wash 3 times with TBS, 5 minutes each time;

[0076] 3) Incubate the NC membrane with the recombinant protein GST-BBM2 described in SEQ ID No.2, the working concentration is 20-200nM or 0.5-5ug / ml, incubate at 37°C for 1 to 2 hours, wash 3 times with TBST and TBS in sequence , 5min each time;

[0077] 4) Add mouse anti-GST monoclonal antibody, dilute the antibody (1:1000) according to the instructions, incubate at 37 degrees for 2 hours, wash 3 times with TBST, 5 minutes each time, then wash 3 times with...

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PUM

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Abstract

The invention provides recombinant protein used for detecting acetylation of histone loci and an application of recombinant protein. An amino acid sequence comprises two or more Bromo structural domain sequences connected in series, wherein the Bromo structural domain sequences are a) amino acid sequences shown in the SEQ ID NO.1; or b) amino acid sequences which are formed by replacing, deleting and / or adding one or more amino acid residues in the amino acid sequences shown in the SEQ ID NO.1 and have the equivalent functions. The recombinant protein has high affinity for H3K14AC, can be used for replacing commercial antibodies for a Western Blot experiment, is high in sensitivity, has no cross-binding phenomenon, is low in anticipated cost and very stable in batch quality and can rapidly realize batch production.

Description

technical field [0001] The invention relates to the technical field of protein modification detection, and more specifically, to a recombinant protein for detecting acetylation of histone sites and its application. Background technique [0002] The molecular anatomy of post-translational modifications that regulate cellular processes and disease progression is one of the main goals of post-genomic biology research. To date, more than 300 post-translational modifications have been discovered. This is an effective way to alter the basic structure and even function of proteins. Modifications on the side chains of lysines demonstrate the enormous complexity of the molecular network. Lysine is an amino acid residue encoded by 15 ribosomes and known to be modifiable. The lysine side chain has two characteristics: rich charge and nucleophilicity, suitable for post-translational modification and covalent bonding with different electrophilic substrates. Many post-translational mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C07K19/00C12N15/70C12N15/11C07K16/00C12N1/21G01N33/68C12R1/19
CPCC07K14/00C07K16/00C07K2319/23C12N15/70G01N33/68
Inventor 李珊珊
Owner HUBEI UNIV