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Method for protein modification

A protein modification and histone technology, applied in the field of protein modification, can solve the problems of expensive materials and cannot meet the needs of protein synthesis, and achieve the effect of shortening the reaction time and reducing the cost.

Pending Publication Date: 2019-11-19
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can obtain relatively pure peptides, the materials for this method are expensive, and it takes more than ten hours to extend each amino acid. Currently, the relatively mature peptides synthesized are less than 100 amino acids, which cannot meet the needs of larger protein synthesis. needs

Method used

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  • Method for protein modification
  • Method for protein modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Thioacetylation modification of the No. 16 lysine site on H4

[0037] With pUC19-UTRG vector containing human histone H4 sequence (such as figure 1 Shown is the pUC19 vector map used to insert the target protein fragment. The DNA fragment site is used to insert any protein fragment) to prepare a DNA template with a lysine codon mutation site, and mutate the codon encoding lysine at position 16 of human histone H4 to an amber terminator (TAG ), as shown in SEQ ID NO:2 (source sequence) and SEQ ID NO:4 (mutated sequence). The specific method is to use the pUC19-UTRG vector containing the H4 fragment as a template to design reverse primers for mutation, and perform PCR amplification. The specific operation refers to Mutan BESTKit (TaKaRa, R401), and the amplified fragments are subjected to agarose gel electrophoresis. (Such as figure 2 As shown, the band marked 2 is the mutant fragment of H4), and purified by gel cutting, the purified fragment was end-ligate...

Embodiment 2

[0040] Example 2. Acetylation modification of lysine 27 on H3.

[0041] Use the pUC19-UTRG vector containing the human histone H3 sequence to prepare a DNA template with a lysine codon mutation site, and mutate the codon encoding lysine at position 27 of human histone H3 to an ocher terminator (TGA), as shown in SEQ ID NO: 1 (source sequence) and SEQ ID NO: 3 (mutated sequence). The specific method is to design a reverse primer for mutation using the pUC19-UTRG vector containing the H3 fragment as a template, and perform PCR amplification. The specific operation refers to the Mutan BEST Kit (TaKaRa, R401), and the amplified fragment is subjected to agarose gel electrophoresis (eg figure 2 As shown, 1 is the amplification product of the H3-K27 template), and purified by gel cutting, and the purified fragments were end-ligated with topoisomerase, and then transferred into E. coli DH5α to obtain mutants. The mutant vector was linearized and concentrated to obtain a purified pr...

Embodiment 3

[0044] Example 3. Simultaneous synthesis of H3-AcK27 and H4-ThioAcK16 in an in vitro expression system.

[0045] The mutants of Example 1 and Example 2 were used as templates.

[0046] Prepare tRNA sep and Flexizyme. For specific operations, refer to Embodiment 1.

[0047] Mix 1μl tRNA (250mM), 1μl Flexizyme (250mM), and 1μl HEPS-KOH (500mM), heat at 95°C for 3 minutes and then cool at room temperature, add 2μl ThioAcK compound (25mM), mix well and ice-bath for 6 hours to get ThioAcK-tRNA, the results were detected by Acid-PAGE.

[0048] Mix 1μl tRNA (250mM), 1μl Flexizyme (250mM), and 1μl HEPS-KOH (500mM), heat at 95°C for 3 minutes and then cool at room temperature, add 2μl AcK compound (25mM), mix well and ice-bath for 6 hours to get ThioAcK-tRNA, the results were detected by Acid-PAGE.

[0049] Using the PURE EXPRESS SYSTEM (NEB, E6800S) in vitro protein expression system, add 1 μL of H3-K27 and H4-K16 templates and 0.5 μL of ThioAcK-tRNA and AcK-tRNA synthesized in t...

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Abstract

The present invention discloses a method for protein modification. The method comprises the following steps: providing a vector comprising a target protein gene sequence, mutating codons encoding amino acids in the target protein gene sequence; constructing unnatural amino acids; respectively preparing Flexizyme and a tRNAsep; using the Flexizyme to ligate the unnatural amino acids to the above tRNAsep to obtain ThioAcX-tRNA; and adding the ThioAcX-tRNA and the codon-mutated target protein to a PURE EXPRESS protein expression system to conduct reaction to obtain a modified protein product. Byadding transfer RNA pre-assembled with the unnatural amino acids in an in vitro protein translation system and using a principle of codon suppression, the method can conduct modification of relativelylarge protein fragments, greatly shortens reaction time, reduces cost, and at the same time can complete synthesis of two or more proteins in the same system.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a protein modification method. Background technique [0002] Protein post-translational modifications (PTMs) include carbohydrates, lipids, nucleic acids, methylation, sulfuric acid, carboxyl, methyl, acetyl, hydroxyl and other functional groups that are covalently linked to proteins. After modification, proteins are endowed with new functions in terms of binding, catalysis, regulation and physical properties. Taking histones as an example, the above-mentioned post-translational modifications will affect the configuration of histones to a certain extent, thereby affecting the formation of nucleosomes, chromosomes, etc. The post-translational modifications of histones directly affect the transcriptional repression and activation of genes. DNA damage, repair, chromosome condensation, recombination and other important life processes. In 2000, the hypothesis of the histone code was pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K14/47
CPCC12P21/02C07K14/47
Inventor 熊海肖秋芸赵烜
Owner SHENZHEN UNIV
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