Protein renaturation method of hydrophobic super-macroporous microsphere synergetic ultrafiltration technology
A super-large pore and hydrophobic technology, applied in the biological field, can solve problems such as membrane fouling and limiting protein renaturation efficiency
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Embodiment 1
[0019] Embodiment 1, the renaturation of lysozyme ultrafiltration that utilizes miniature ultrafiltration device to carry out
[0020] 1. Weigh 20mg of lysozyme and dissolve it in 20mL denaturation solution (100mmol / L Tris-HCl+1mmol / L EDTA+30mmol / L DTT+8mol / L urea (pH 8.5)), so that the final concentration of lysozyme is 1mg / L mL. Keep the solution at 37°C for 4 hours to completely denature the lysozyme;
[0021] 2. Cut a circular ultrafiltration membrane with a diameter of 24mm from a circular ultrafiltration membrane with a diameter of 90mm (molecular weight cut-off of 10kDa, made of polyethersulfone) purchased from Pall Corporation of the United States, and place it in the refolding buffer solution (100mmol / L Tris-HCl+1mmol / L EDTA+1mmol / L GSSG+10mmol / L GSH+2mol / L urea (pH 8.5)) overnight, and then put it into a micro-ultrafiltration refolding device with a volume of 16mL (See the attached schematic diagram of the device figure 2 );
[0022] 3. Inject 16 mL of denatured...
Embodiment 2
[0024] Embodiment 2, utilize 0.5m 2 Ultrafiltration renaturation of lysozyme by plate ultrafiltration device
[0025] 1. Weigh 150mg of lysozyme and dissolve it in 300mL denaturation solution (100mmol / L Tris-HCl+1mmol / L EDTA+30mmol / L DTT+8mol / L urea (pH 8.5)), so that the final concentration of lysozyme is 0.5mg / mL. Keep the solution at 37°C for 4 hours to completely denature the lysozyme;
[0026] 2. Adopt the Pellicon II system of U.S. Millipore Company to carry out ultrafiltration renaturation (see attached image 3 ). Set the membrane area to 0.5m 2 The polyethersulfone membrane package (molecular weight cut-off is 10kDa) is fixed in the ultrafiltration device, and the refolding buffer solution (100mmol / L Tris-HCl+1mmol / L EDTA+1mmol / L GSSG+10mmol / L GSH+2mol / L Urea (pH8.5)) washes the membrane bag;
[0027] 3. Inject 300mL of denatured lysozyme solution into the above-mentioned plate-type ultrafiltration refolding device, add hydrophobic super-large porous microspher...
Embodiment 3
[0029] Embodiment 3, utilize the ultrafiltration renaturation of the nitrilase that utilizes miniature ultrafiltration device to carry out
[0030] 1. Weigh 4 mg of nitrilase and dissolve it in 20 mL of denaturing solution (50 mmol / L Tris-HCl+10 mmol / L DTT+8 mol / L urea (pH 8.5)) to make the final concentration of nitrilase 0.2 mg / mL. Keep the solution at 4°C for 12 hours to completely denature the nitrilase;
[0031]2. Cut a circular ultrafiltration membrane with a diameter of 24mm from a circular ultrafiltration membrane with a diameter of 90mm (molecular weight cut-off of 50kDa, made of polyethersulfone) purchased from Pall Corporation of the United States, and place it in the refolding buffer solution (50mmol / L Tris-HCl + 1.3mmol / L EDTA + 1.3mmol / L GSSG + 1.33mmol / L GSH + 1mol / L urea (pH 8.5)) overnight, and then put into a micro ultrafiltration complex with a volume of 16mL sex device (see attached figure 2 );
[0032] 3. Inject 16 mL of denatured nitrilase solution in...
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