Biology Reference
In-Depth Information
24.2.2.1.3
Affinity chromatography
At the end of the incubation, centrifuge the homogenate at 50,000
g
for 30 min to re-
move insolublematerial. Add 5 mLof alprenolol-agarose gel to the supernatant and rotate
at 4
C for binding overnight. Next day, recover the alprenolol-agarose gel by pouring it
into an empty wide gravitation column and wash the bound protein extensively with
washing buffer (150-200 mL of 50 mM HEPES, pH 7.5, 0.15 M NaCl, 0.05% DDM,
and 0.01% CHS). Elute the bound receptor batchwise by competition with 2 mM alpre-
nolol in washing buffer (twice with 10 mL and once finally with 5 mL) over a total period
of 4-5 h of slow rotation. Check the purity of eluted fractions by SDS-PAGE.
Incubate the eluted protein (25 mL) with
L of nickel affinity gel and ro-
tate for at least 2 h at 4
C. Then, wash the nickel affinity gel extensively with
150 mL of washing buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.025%
DDM, and 0.005% CHS containing 50
500
m
M carazolol). (This step can also be used
for ligand exchange.) Perform elution of the sample at the end of incubation by com-
petition with 1-2 mL of 200 mM imidazole in the washing buffer. Further purify the
eluted samples by gel filtration in 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.025%
DDM, and 0.005% CHS containing 50
m
M carazolol. Concentrate the peak fractions
from gel filtration (typically 1-2 mL) up to 50 mg/mL for crystallization. A typical
yield from 3 L of culture for the
m
b
2-AR is 3-4 mg protein (
Fig. 24.4
A).
FIGURE 24.4
Crystal structure of
2-AR(E122W)/
T4L sample developed with silver staining. Both the purified monomer and dimer are present.
(B, C) Representative crystals imaged under crossed polarizers. Inset in panel B shows a
close-up of microcrystals. (B) Crystals grown in 0.1 M HEPES, pH 7.0, 0.15 M ammonium
fluoride, 30% PEG 400, and 7% 1,4-butanediol. (C) Crystals grown in 0.1 M HEPES, pH 7.0,
0.15 M sodium sulfate, 30% PEG 400, and 7% 1,4-butanediol. (D) Representative X-ray
diffraction pattern from a microcrystal with dimensions of 20
b
2-AR(E122W)/T4L. (A) SDS-PAGE analysis of a purified
b
10
5
m
m. (E) A part of
electron density of
b
2-AR(E122W)/T4L crystals.
