Chromatography freezers in restructuring the modification application of phospholipase C, whose character is: includes the following steps:
1, modifier SS ‑ mPEG preparation: to add 2 g flask molecular weight is 5000 polyethylene glycol monomethyl oxygen radicals mPEG, add 4 MLN, N ‑ dimethyl formamide, fully dissolved;
Then add 0.
12 g succinic anhydride, sealed blending, at 100 ℃, stirring and heating 3 h;
By drops in the reactant chromatography freezer after cooling, add 10 ml ether, proceeds to precipitate filter dry;
After drying the precipitate with methylene chloride compound solution, reoccupy ether precipitation, repeat four times.
The precipitation product vacuum drying, kept in a dryer spare;
Will the end product soluble in 4 MLN, N ‑ dimethyl formamide, adding 0.
46 designed. the gn ‑ hydroxy succinimide and 0.
825 g dicyclohexyl carbon 2 imide, 30 ℃ dissolve overnight, the reaction product put in chromatography of air-conditioner cooling to 0 ℃ and 40 ml ether by drops to join, to filter the precipitation dry;
After drying the precipitate with methylene chloride compound solution, reoccupy ether precipitation, repeat four times.
Is the activation of the polyethylene glycol precipitation products, it is dry, alternate;
2, modification enzyme preparation: the SS ‑ mPEG and phospholipase C in the molar ratio of 0 ~ 30 dissolved for 10 ml volume, pH of 7.
2, the concentration of 25 mm Tris ‑ HCl buffer, shock response time at room temperature, with the dialysis bag 4 ℃ dialysis for the night, the modification enzyme surface dry cold backup.
