| Recovery of rhG-CSF inclusion bodies was followed23 by some modifications. The cells were thawed at room temperature and cleaned up with 0.020 mol · L-1 Tirs-HCl (pH 8.0), and then the suspension was centrifuged at 7,000 rpm and 4°C for 10 min after washing. The supernatant was discarded. After being frozen at -20°C for 12 h, 100 g of the frozen cells were thawed at room temperature and resuspended in 1000 mL of 0.050 mol · L-1 Tris-HCl buffer (pH 8.0) containing 1.0 m mol · L-1 EDTA. The cells were lysed by sonication in an ice water bath. The lysates were centrifuged at 14,000 rpm for 20 min to collect the insoluble protein aggregates. The pellet (protein aggregates and cell debris) was washed with 500 mL of the following solutions, 0.020 mol · L-1 Tris-HCl (pH 8.0) containing 0.010 mol · L-1 EDTA and 2.0 mmol · L-1 -mercaptoethanol (-ME), 0.020 mol · L-1 Tris-HCl (pH 8.0) containing 2.0 mol · L-1 urea and 2.0 mmol · L-1 EDTA, 0.020 mol · L-1 Tris-HCl (pH 8.0) containing 70% 2-propanol, respectively. Finally, the inclusion bodies were washed with 0.02 mol · L-1 Tris-HCl (pH 8.0). After each washing step, the suspension was centrifuged at 14,000 rpm and 4°C for 15 min and the supernatant was discarded. About 18.0 g of pellet fraction containing rhG-CSF inclusion bodies were obtained and stored at -20°C.
Solubilization of rhG-CSF from Inclusion Bodies
Solubilization of rhG-CSF was also followed23 by some modification. Purified inclusion bodies (4.0 g) was solubilized in 20 mL of 7.0 mol · L-1 GuHCl, 1.0 mmol · L-1 EDTA, 100 mmol · L-1 -ME, 50 mmol · L-1 Tris (pH 8.0) with stirring at room temperature for 1 h. The suspension was centrifuged at 14,000 rpm for 15 min to remove insoluble debris and the supernatant was diluted by 20 mmol · L-1 Tris (pH 8.0), 1.0 mmol · L-1 EDTA to the final concentration of GuHCl to 5.0 mol · L-1. After centrifugation, the solution was diluted again by 20 mmol · L-1 Tris (pH 8.0), 1.0 mmol · L-1 EDTA to the final concentration of GuHCl to 2.0 mol · L-1. Then the precipitated protein consisting mainly of rhG-CSF was dissolved in 8.0 mol · L-1 urea, 1.0 mmol · L-1 EDTA, 100 mmol · L-1 -ME, 50 mmol · L-1 Tris (pH 8.0). The suspension stood for 12 h at room temperature with continuous stirring. After centrifugation, the supernatant containing rhG-CSF was collected.
Procedures for the Refolding of rhG-CSF by SEC
A chromatographic run was carried out at room temperature using a SEC column (20×2.6 cm I.D.) packed with Superdex 75. The SEC column was equilibrated with mobile phase containing 0.15 mol · L-1 sodium chloride, 0.1 mol · L-1 Tris (pH 8.0), 1.0 mmol · L-1 EDTA, 2.5 mmol · L-1 GSH, 0.8 mmol · L-1 GSSG, 3.0 mol · L-1 urea, 15% glycerol (v/v). The solubilized and denatured rhG-CSF (200 µL) in 8.0 mol · L-1 urea solution was injected directly into the column. The renatured rhG-CSF was eluted with the same mobile phase as used for equilibration at a flow rate of 2.0 mL · min-1. Detection was set at 280 nm. The fractions containing rhG-CSF were collected and was adjusted to pH 4.0 by hydrochloric acid and dialyzed against a storage solution containing 10.0 mmol · L-1 sodium acetate buffer at pH 4.0. The solution containing rhG-CSF was used for determination of protein concentration and bioactivity. |