Recombinant type II restriction endonucleases, MmeI and related endonucleases and methods for producing the same
An endonuclease, restriction enzyme technology, applied in Ming
According to the field of the present invention, can solve the problems such as not being used
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Embodiment 1
[0050] Purification of endonuclease MmeI
[0051] A single clone of methanolophilic bacteria (NEB#1190) was grown in 1 liter medium M (0.08 μM CuSO 4 , 0.448μM MnSO 4 , 0.348μM ZnSO 4 , 6.0μM FeCl 3 , 18μM CaCO 3 , 1.6mM MgSO 4 , 9.0mM NaH 2 PO 4 , 10.9mM K 2 HPO 4 , 13.6mM (NH 4) 2 SO 4 ) for 24 hours. Use this culture to inoculate 100 liters of medium M. These cells were cultured overnight at 37°C under aerobic conditions until stationary phase. 752 grams of wet cell pellet were collected from five 100-liter fractions of fermentation broth.
[0052] Suspend 750 g of the Methanotroph cell pellet in 2.25 L of Buffer A (20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1.0 mM DTT, 0.1 mM EDTA, 5% Glycerol) and homogenize by Gaulin at approximately 12,000 psig The supernatant was collected after the lysate was centrifuged at 13000×G for 40 minutes.
[0053] The supernatant solution was applied to a 500 ml Heparin Hyper-D column (Biosepra SA) which had been equilibrated with b...
Embodiment II
[0066] Cloning of MmeI endonuclease
[0067] 1. DNA purification: preparation of total genomic DNA of methanolophilic bacteria. 5 g of sedimented cells were suspended in 20 ml of 25% sucrose, 0.05 M Tris-HCl pH 8.0, to which 10 ml of 0.25 M EDTA, pH 8.0 was added. Then 6 ml of lysozyme solution (10 mg / ml lysozyme in 0.25 M Tris-HCl, pH 8.0) was added, after which the cell suspension was left at 4° C. for 16 hours. Next, 25 ml of lysis mixture (1% Triton-X100, 0.05M Tris, 62 mM EDTA, pH 8.0) and 5 ml of 10% SDS were added, followed by standing at 37°C for 5 minutes. Then use one volume of equilibrium phenol: chloroform: isoamyl alcohol (50:48:2, v / v / v) to extract the solution, recover the aqueous phase, and then use one volume of chloroform: isoamyl alcohol (24:1 , v / v) extracted twice. The resulting aqueous solution was dialyzed four times against 2 liters of 10 mM Tris, 1 mM EDTA, pH 8.0. The dialyzed DNA solution was digested with RNase (100 µg / ml) at 37°C for 1 hour. D...
Embodiment III
[0191] MmeI endonuclease provides protection against MmeI cleavage under in vivo conditions
[0192] Plasmid pTBMmeI.1 was purified from NEB1457 by Qiagen's miniprep procedure. This plasmid has two MmeI sites in the vector backbone and one MmeI site in the MmeI gene. The plasmid was digested with MmeI to test whether this DNA is resistant to MmeI endonuclease activity, this resistance means that a single MmeI gene can methylate DNA under in vivo conditions to protect host DNA from endonuclease activity . For detection mix the following reagents:
[0193] 10 μl pTBMmeI.1 miniprep DNA
[0194] 15 μl 10×NEBuffer4
[0195] 15 μl SAM (1 mM stock solution)
[0196] 110 μl dH 2 o
[0197] 1 μl MmeI endonuclease (15 units).
[0198] The reaction mixture was divided into three parts after mixing. Add 0.5 μl water to the first one-third reaction, add 0.5 μl pRRS vector to the second one-third reaction, add 0.5 μl PhiX174 DNA to the third one-third reaction as positive control....
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