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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 -charged
HisTrap chelating column (HT; lane 7), and gel-filtration pool (GF; lane 8). MW, molecular
weight markers (in kDa).
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and RXRa LBD
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