DNA methyltransferase and its soluble heterologous expression and separation and purification method

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

Active Publication Date: 2020-07-24
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 methyltransferase and its soluble heterologous expression and separation and purification method
  • DNA methyltransferase and its soluble heterologous expression and separation and purification method
  • DNA methyltransferase and its soluble heterologous expression and separation and purification method

Examples

Experimental program
Comparison scheme
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 a variety of expression vectors (including pET-28a, pET-22b, pRRS and pET28a-HMT, etc.), hosts (including BL21, BL21 (DE3), BL21 (DE3), pLysS, Transetta, etc.) (DE3), TransB (DE3) and E.coli ER2566, etc.) and induced expression conditions (such as IPTG concentration, induction temperature and time, etc.), and finally select pET28a-HMT with good soluble expression and E.coli The ER2566 host is used as a prokaryotic expression system (see Table 1).

[0027] (1) Use Tiangen Biotech's bacterial genomic DNA extraction kit (DP302-02) to extract genomic DNA of Deinococcus radiodurans, and NANODROP 1000 (Thermo, USA) to determine DNA concentration and purity. Design a pair of full-length specific primers for the DNA methyltransferase M.DraR1 coding gene sequence, and introduce the same restricti...

Embodiment 2

[0037] Example 2: Prokaryotic expression of DNA methyltransferase M.DraR1

[0038] (1) Escherichia coli E.coli Preparation of ER2566 competent cells: E.coli After the ER2566 strain (ZonHonBiopharma Institute, Inc., ZHR5015) was activated by streaking on a non-resistant LB solid petri dish, a single clone was picked and inoculated into 5 mL of LB liquid medium and cultured overnight at 37°C and 220 rpm with shaking. ER2566 competent cells were prepared aseptically according to the competent cell preparation method in the "Molecular Cloning Guide", 100 μL aliquoted and stored in an ultra-low temperature refrigerator at -80 ℃ for later use.

[0039] (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 gently, and ice bath for 30 min , 42 ℃ water bath heat shock for 45-90 s, and then ice bath for 2-3 min, add 500 μL of steril...

Embodiment 3

[0043] Example 3: Separation and purification of DNA methyltransferase M.DraR1

[0044] (1) According to the method of Example 2(3) and Example 2(4), the target protein was induced and cultured. After the induction, the bacteria were collected by centrifugation at 8000 rpm, 4°C, 10 min, washed with 1×PBS solution once, and centrifuged again to collect Bacteria, store at -80 ℃ for later use. Add 20 mL of Lysis Buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 3 mM β-Me, 5% Glycerol, 9 mM Imidazole) to suspend cells at a ratio of 20:1, that is, 1 g of bacteria (wet weight). Cool in an ice bath for 5-10 minutes.

[0045] (2) Cell disruption: The above suspended cells are first crushed by a high-pressure homogenizer (Shanghai Litu, FB-110 series), the parameters are 4 ℃, 800-1200 bar, and time 2-4 min. Then continue to use ice water bath to ultrasonically disrupt the cells (Ningbo Xinzhi, JY92-IIN), the parameters are 60% power, ultrasonic 3s, gap 9.9s, and time 60-90 min. After sonicatio...

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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 method for soluble heterologous expression and separation and purification 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 enzyme that mediates and catalyzes DNA methylation modification is mainly DNA methylation transferase, which is widely distributed in all prokaryotic and eukaryotic organisms, and can specifically recognize and modify specific base positions in specific sequences of the genome. In almost all bacteria or prokaryotes, DNA methyltransferase and restriction endonucleases that recognize the same site constitute the primary immune defense system of bacteria—restriction modification system, which protects host cells from foreign genome invasion, thereby maintain...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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