Table 1.

Recombinant Human Apolipoprotein Purification Protocol

Step	Parameter	Value	Description
1	Centrifugation		15,000 rpm, 10 min
2	Dilution	4 times	20 mM NaAc–HAc buffer
3	Adjusted pH	3.0	HAc
4	Cation exchange chromatography	0.5 mL/min	To bind rhApoA-II
5	Elution	1 mL/min	A linear gradient of NaCl
6	Collected eluted proteins		Analysis of eluted proteins
7	Reverse phase chromatography	0.5 mL/min	Desalination and further purification
8	Elution	1 mL/min	Methanol
9	Collected eluted proteins		Analysis of eluted proteins
10	Remove D methanol		Vacuum distillation and freeze drying

Step Notes

1. Collected supernatant form fermentation broth.

2. The supernatant was diluted four times with 20 mM NaAc–HAc (pH 3.0) buffer.

3. The pH of the supernatant was adjusted to 3.0 with 1 M acetate acid.

4. The supernatant was loaded onto the cation exchange chromatographic column at the rate of 0.5 mL/min.

5. The bound protein was eluted with a linear gradient of 0.1–1.0 M NaCl, while the flow rate was maintained at the rate of 1 mL/min.

6. Protein elution was monitored by measuring the absorbance at 280 nm and SDS-PAGE analysis.

7. Column eluent containing rhApoA-II was loaded onto a reverse phase column, which was equilibrated with 0.1% TFA, for the further purification.

8. RhApoA-II was eluted using 50% and 100% methanol (containing 0.1% TFA) at the rate of 1 mL/min.

9. Protein elution was monitored by measuring the absorbance at 280 nm and SDS-PAGE analysis.

10. RhApoA-II was concentrated by vacuum distillation and freeze drying to remove methanol.

rhApoA-II, recombinant human apolipoprotein A-II; TFA, trifluoroacetic acid; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.