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.
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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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