Method for preparing recombinant human insulin crystal
A technology of recombinant human insulin and insulin, which is applied in the field of protein crystallization to achieve the effect of short freeze-drying time, large particles and fast sedimentation speed
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Embodiment 1
[0043] 1) Add zinc chloride solution to the reverse high-efficiency chromatography solution containing recombinant human insulin, so that every OD 280 nm of recombinant human insulin makes each OD 280 nm recombinant human insulin contains zinc ions at a concentration of 14 mg / ml, add low-temperature purified water, and when the concentration of the insulin solution is diluted to 4 mg / ml, turn on the stirrer switch and stir;
[0044]2) Add 3-5M ammonia water to the insulin solution in step 1) to adjust the precipitation to promote precipitation until the pH is between 7.0, stop stirring; transfer the primary crystallization solution to a crystallization tank and let it settle. , Determination of supernatant OD 280 When nm is less than or equal to 0.1, remove the supernatant and obtain a crystal;
[0045] 3) Transfer the first knot crystal to the second knot settling tank, add 1-3M acetic acid to the second knot settling tank to dissolve, turn on the stirrer, and stop adding a...
Embodiment 2
[0054] 1) Add zinc chloride solution to the reverse high-efficiency chromatography solution containing recombinant human insulin, so that every OD 280 nm of recombinant human insulin makes each OD 280 nm recombinant human insulin contains zinc ions at a concentration of 46 mg / ml, add low-temperature purified water, and when the concentration of the insulin solution is diluted to 0.5 mg / ml, turn on the stirrer switch and stir;
[0055] 2) Add 3-5M ammonia water to the insulin solution in step 1) to adjust the precipitation and promote precipitation until the pH is between 5.5, stop stirring; transfer the primary crystallization solution to a crystallization tank and let it settle. , Determination of supernatant OD 280 When nm is less than or equal to 0.1, remove the supernatant and obtain a crystal;
[0056] 3) Transfer the first knot crystal to the second knot settling tank, add 1-3M acetic acid to the second knot settling tank to dissolve, turn on the stirrer, and stop stir...
Embodiment 3
[0064] 1) Add zinc chloride solution to the reverse high-efficiency chromatography solution containing recombinant human insulin, so that every OD 280 nm of recombinant human insulin makes each OD 280 nm recombinant human insulin contains zinc ions at a concentration of 55 mg / ml, add low-temperature purified water, and when the concentration of the insulin solution is diluted to 6 mg / ml, turn on the stirrer switch and stir;
[0065] 2) Add 3-5M ammonia water to the insulin solution in step 1) to adjust the precipitation to promote precipitation until the pH is between 7.5, stop stirring; transfer the primary crystallization solution to a crystallization tank and let it settle. , Determination of supernatant OD 280 When nm is less than or equal to 0.1, remove the supernatant and obtain a crystal;
[0066] 3) Transfer the first knot crystal to the second knot settling tank, add 1-3M acetic acid to the second knot settling tank to dissolve, turn on the stirrer, and stop stirrin...
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