Advertisement
h of induction at 30
"C,
cells were h m c s l e d by centrifugation at 3.000 g for 10 min.
1 OC m d frozen at
-
70 "C.
3.2.3.3.5. Purification of recombinant rhitinase
A11 steps were carried out at 4°C unless specified. After two freeze-thaw
cycles.
E. coli
cells harvested were resuspended in lysis buffer (50 mM Tris HCI, pH
7.8. 1 mM EDTA) and homogenized by sonication (50% duty cycle. I min.; output
control setting 5) using a 0.75 inch (approx. 1.9 cm) tip (model. 250. 20
kHz
Sonitier;
Brnnson Power Company, Danbury, CT, U.S.A) for a total of 20 min., with pulse and
interval time of 1 min. and 30 s respectively for each duty cycle. The mixture was
centrifuged at 13,500
g
for 10 min. at 4°C to remove the pellet and the supernatant.
The pellet containing inclusion bodies after sonication was resuspended in
wash buffer ( I 0 mM Tris HCI pH 7.5. 300 mM NaCI. I mM EDTA. 1% Triton X-
100
and
I
M urea). Washing was done 5 times at 12,000 g for 10 min. at 4 OC. follvwed
by extensive washing with sterile double distilled water to remove EDTA. T o renature
the protein properly from inclusion bodies the pellet was dissolved in 10X volume of
denaturation-renaturation (DR) buffer (50 mM Tris HCI. (pH 7.8). 5 mM MgC12, 2.5
mM B-mercaptoethanol, 0.1% (wlv) Tween 20) containing
8
M urea and incubated at
room temperature for 1 h. The mixture was centrifuged at 10,000 g for 20 min. at
4
"C. The supernatant was dialyzed gradually against DR buffer containing
Progressively low concentrations
(6,
5, 4, 2 and I mM) of urea, dialyzed overnight in
DR buffer and in sterile distilled water. The dialyzed protein was concentrated by
l~ophilization.
Advertisement
