| Expression was conducted in our institute according to the
reference [21]. Cultures were grown at 37 ?C in LB medium
supplemented with kanamycin (100 g/mL) in 5 L fermentor,
and induced at a cell density of 0.5 (A600 nm) by the
addition of 0.001 mol/L isopropyl-thiogalactoside (IPTG).
Once cells reached an optical density of 1.2, they were harvestedby centrifugation at 6000 rpm and 4 ?C for 15 min,
and frozen at ?20 ?C. Forty-two grams of wet biomass were
obtained finally.
The cells were thawed at room temperature and washed
with a buffer of sodium phosphate (0.02 mol/L, pH 7.2)
containing 0.001 mol/L EDTA, and then the suspension
was centrifuged at 6000 rpm and 4 ?C for 15 min after
washing. The supernatant was discarded, and this procedure
was repeated once. The cells were frozen at ?20 ?C
for 24 h. Twenty grams of the frozen cells were thawed at
room temperature and resuspended in 200mL of a cold
buffer of 0.02 mol/L sodium phosphate (pH 7.2) containing
0.001 mol/L EDTA. The cells were lysed by sonication on
ice. The lysates were centrifuged at 18,000 rpm for 15 min
to collect the insoluble protein aggregates. The pellet (protein ggregates and cell debris) was washed twice with
30mL of 0.1 mol/L Tris–HCl (pH 8.0) including 4.0 mol/L
urea, 0.2 mol/L NaCl and 0.02 mol/L EDTA. The pellet as subsequently washed with 30mL of 1.0 mol/L NaCl. fter each washing step, the suspension was centrifuged t 18,000 rpm and 4 ?C for 15 min. About 2.4 g of pellet raction containing PrP(104?42) inclusion bodies was btained and stored at ?20 ?C prior to further separation.
After thawing, 1.0 g of PrP(104?42) inclusion bodies
were resuspended in 20mL of solubilization buffer composed
of 8.0 mol/L urea, 0.1 mol/L Tris–HCl (pH 8.0),
0.2 mol/L NaCl, 0.1 mol/L -mercaptoethanol, 20 mmol/L
EDTA. After overnight incubation at 4 ?C, the solution was
centrifuged at 18,000 rpm and 4 ?C for 15 min, the supernatant ontaining PrP(104?42) was stored at 4 ?C. The total protein concentration in the supernatant was determined to be 8.0 mg/mL by BCA Protein Assay Kit. The purity of PrP(104?42) in the inclusion body was measured to be 14% by SDS–PAGE and thin-layer chromatographic
scanner. Through calculation, about 115 mg of recombinant
PrP(104?42) could be obtained from the expression
system.
PrP(104?42) was purified by HPHIC on a prepacked column.
The column liquid chromatography was performed at
room temperature on a HPLC system. The PrP(104?42) extract
was centrifuged without further treatment at 18,000 rpm
and 4 ?C for 15 min prior to sample loading. One millilitre
of the sample solution containing 8.1 mg of total protein was
directly injected into the HPHIC column previously equilibrated
with 80% solution A (3.0 mol/L ammonium sulfate
containing 0.05 mol/L phosphate, pH 7.0) and 20% solution
B (0.05 mol/L phosphate, pH 7.0). The protein desorption
was performed using a nonlinear gradient elution in 40 min
as in the Fig. 1c. HPHIC flow rate was 1.0 mL/min at UV
detection was set at 280 nm. Protein extracts were separated
by SDS–PAGE. Appropriate fractions were pooled and their
protein concentrations were estimated by the BCA Protein
Assay Kit. |