Table 1.
Protocol Table for Recombinant Human High-Density Lipoprotein Preparation and Purification
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Phosphate buffer | 12 mL | pH 7.4 |
| 2 | Egg phosphatidylcholine and cholesterol | 0.5 mL | Dissolved in ethanol |
| 3 | Mixing step | 15 min | Under a stream of N2 |
| 4 | Six kinds of rhapo | 1 mL | Dissolved in phosphate buffer |
| 5 | Incubation time | 30 min | Room temperature |
| 6 | Incubation time | 12 h | 4°C |
| 7 | Dialysis | 2 days | 4°C, against phosphate buffer |
| 8 | Density-gradient centrifugation | 20 h | As desired |
Step Notes
1. 50-mL centrifuge tube, 12 mL phosphate buffer, pH 7.4.
2. An ethanol solution containing 7.2 mg egg phosphatidylcholine and 1.6 mg cholesterol, rapidly injected into phosphate buffer.
3. Mixing for 15 min under a stream of N2.
4. Six kinds of rhapo were dissolved in phosphate buffer (1 mL) as their proportion in native. HDL and added to the lipid mixture with stirring.
5. Mixture was incubated for 30 min at room temperature.
6. Mixture was incubated at 4°C for 12 h.
7. Solution was dialyzed totally at 4°C (about 2 days) against phosphate buffer to remove ethanol and cholate.
8. A discontinuous NaCl/KBr density gradient was used to purify rhHDL.
rhHDL, recombinant human high-density lipoprotein; rhapo, recombinant human apolipoprotein.