Biology Reference
In-Depth Information
to both isolated LBDs, which contain the major dimerization function, and
full-length receptors. In the succeeding text, we provide the example of a rapid
two-step copurification procedure yielding nonaggregated and functionally homo-
geneous RAR
a
-RXR
a
LBD heterodimers in large quantities suitable for structural
and other
in vitro
studies. The purification strategy is based on the observation that
RAR
a
is always isolated as a monomer, whereas RXR
a
exists as a mixture of
monomers, homodimers, and homotetramers (
Chen et al., 1998
). Therefore, a
histidine tag is added to RAR
a
(His-RAR
a
) only but not to RXR
a
. Upon mixing
the extracts of
Escherichia coli
programmed to express His-RAR
a
and RXR
a
,
His-RAR
a
-RXR
a
heterodimers are formed. In the subsequent immobilized metal
affinity chromatography, only His-RAR
a
monomers and His-RAR
a
-RXR
a
heterodimers are recovered, while excess of RXR
a
does not bind to the nickel
resin. Next, the excess of His-RAR
a
monomers and aggregated proteins is easily
removed by a gel-filtration step. The results of a typical purification procedure are
shown in
Fig. 2.3
.
2.1.2.1
Required materials
- pET-15b and pET-3a expression vectors (Novagen)
- Competent
E. coli
BL21(DE3) cells (Novagen)
- Shaker/incubator set at 37
C
- Sonicator
- Liquid chromatography system (AKTA purifier, GE Healthcare)
- HisTrap chelating columns (GE Healthcare)
- HiLoad 26/60 Superdex 75 column (GE Healthcare)
- UV-Vis spectrophotometer (NanoDrop, Thermo Scientific)
FIGURE 2.3
Generic purification of RARa-RXRa LBD heterodimers. Representative SDS-polyacrylamide
gel of noninduced and IPTG-induced bacteria (RARa LBD
; lanes
2-5), crude extract (CE; lane 6), pool of the heterodimer eluted from the Ni
2þ
-charged
HisTrap chelating column (HT; lane 7), and gel-filtration pool (GF; lane 8). MW, molecular
weight markers (in kDa).
þ
/
and RXRa LBD
þ
/