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DNA (deoxyribonucleic acid) methyltransferase, and soluble heterologous expression method and isolation and purification method for DNA methyltransferase

A methyltransferase, separation and purification technology, applied in the field of DNA methyltransferase and its soluble heterologous expression and separation and purification, can solve the problem of inability to obtain purified protein, in vitro expression, DNA methyltransferase expression and purification Difficulties in the process, etc.

Active Publication Date: 2019-03-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since Deinococcus radiodurans is a microorganism with strong resistance to extreme conditions such as radiation and drought, many protein activities in its body require unique physiological conditions, so the expression and purification process of DNA methyltransferase in this bacterium is of great importance. It is particularly difficult and requires particularly hard work and wisdom
We have tried a large number of reported DNA methyltransferase expression and purification methods, none of which can be expressed in vitro, or exist in the form of inclusion bodies, and cannot obtain active purified proteins

Method used

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  • DNA (deoxyribonucleic acid) methyltransferase, and soluble heterologous expression method and isolation and purification method for DNA methyltransferase
  • DNA (deoxyribonucleic acid) methyltransferase, and soluble heterologous expression method and isolation and purification method for DNA methyltransferase
  • DNA (deoxyribonucleic acid) methyltransferase, and soluble heterologous expression method and isolation and purification method for DNA methyltransferase

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Cloning of DNA methyltransferase M.DraR1 and construction of expression vector

[0026] In order to obtain soluble target protein, the present invention optimized and screened various expression vectors (including pET-28a, pET-22b, pRRS and pET28a-HMT, etc.), hosts (including BL21, BL21(DE3), BL21(DE3) pLysS, Transetta (DE3), TransB (DE3) and E. coli ER2566, etc.) and induction expression conditions (such as IPTG concentration, induction temperature and time, etc.), and finally select soluble well-expressed pET28a-HMT and E. coli The ER2566 host was used as a prokaryotic expression system (see Table 1).

[0027] (1) Genomic DNA of Deinococcus radiodurans was extracted using the Bacterial Genomic DNA Extraction Kit (DP302-02) of Tiangen Biotechnology, and the DNA concentration and purity were determined by NANODROP 1000 (Thermo Company, USA). Design a pair of full-length specific primers for the coding gene sequence of DNA methyltransferase M.DraR1, and ...

Embodiment 2

[0036] Example 2: Prokaryotic expression of DNA methyltransferase M.DraR1

[0037] (1) Escherichia coli E. coli Preparation of ER2566 competent cells: the E. coli After the ER2566 strain (ZonHon Biopharma Institute, Inc., ZHR5015) was activated by streaking on LB solid culture dishes without any resistance, a single clone was picked and inoculated in 5 mL LB liquid medium, and cultured overnight at 37 °C with shaking at 220 rpm. ER2566 competent cells were aseptically prepared according to the competent cell preparation method in the "Molecular Cloning Guide", 100 μL was aliquoted and stored in a -80 °C ultra-low temperature refrigerator for future use.

[0038] (2) Transformation of pET28a-HMT-M.DraR1 recombinant vector: Take ER2566 competent cells from -80 ℃ ultra-low temperature refrigerator and thaw on ice, add 10-20 μg recombinant expression vector aseptically, mix lightly, and immerse in ice for 30 min , 42°C water bath heat shock for 45-90 s, then ice bath for 2-3...

Embodiment 3

[0042] Embodiment 3: Separation and purification of DNA methyltransferase M.DraR1

[0043] (1) Induce and cultivate the target protein according to the methods in Example 2 (3) and Example 2 (4). After the induction, collect the bacteria by centrifugation at 8000 rpm, 4 °C, 10 min, wash once with 1×PBS solution, and collect by centrifugation again Bacteria were stored at -80°C for future use. Add 20 mL lysis buffer (20 mM Tris-HCl pH8.0, 500 mM NaCl, 3 mM β-Me, 5% Glycerol, 9 mM Imidazole) to suspend cells at a ratio of 20:1. , cooled in an ice bath for 5-10 min.

[0044] (2) Cell crushing: The above suspended cells were first crushed by a high-pressure homogenizer (Shanghai Litu, FB-110 series) with parameters of 4 °C, 800-1200 bar, and 2-4 min. Continue to ultrasonically disrupt the cells in an ice-water bath (Ningbo Xinzhi, JY92-IIN), with the parameters of 60% power, 3 s ultrasonic, 9.9 s interval, and 60-90 min time. After the sonication, 4 ℃, 15000rpm, 30min high-spee...

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Abstract

The invention discloses DNA (deoxyribonucleic acid) methyltransferase, and a soluble heterologous expression method and an isolation and purification method for the DNA methyltransferase. The DNA methyltransferase, the soluble heterologous expression method and the isolation and purification method have the advantages that the N4-Cytosine DNA methyltransferase M.DraR1 which has the DNA methyltransferase activity and is soluble in buffer solution is isolated and purified from deinococcus radiodurans for the first time; nucleotide sequences of the DNA methyltransferase, and the clone and expression method and the separation and purification method for the DNA methyltransferase are further provided; methodological reference can be provided to development and application of other DNA methyltransferase or restriction incision enzymes by the clone and expression method and the isolation and purification method, and the clone and expression method and the isolation and purification method have important guiding significance in developing novel molecular biological tool enzymes.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a DNA methyltransferase and a soluble heterologous expression and separation and purification method thereof. Background technique [0002] DNA methylation is a widespread and very important DNA epigenetic mechanism, which plays an important role in regulating gene expression, genome stability and cell differentiation. The enzymes that catalyze DNA methylation modification are mainly DNA methyltransferases, which are widely distributed in all prokaryotic and eukaryotic organisms, and can specifically recognize and modify specific base positions in specific genome sequences. In almost all bacteria or prokaryotes, DNA methyltransferases and restriction endonucleases that recognize the same site constitute the primary immune defense system of bacteria—restriction modification system, which protects host cells from the invasion of foreign genomes, thereby maintaining Own lif...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12R1/19
CPCC12N9/1007C12N15/70C12Y201/01113
Inventor 华跃进李胜杰王梁燕蔡建玲
Owner ZHEJIANG UNIV
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