Biology Reference
In-Depth Information
(lysis—low salt in
Table 3.1
), for 30 min. The combined action of defrosting, osmo-
lysis, and detergent (Triton) lyses the plasma membrane and releases cytoplasmic
content. Freeze-thawing cycles cannot be employed as DCX degrades. A number
of inhibitors are used to limit the action of a broad spectrum of proteases, and
b
-mercaptoethanol is indispensible to increase the solubility of DCX, which contains
nine cysteines.
To extract DCX from large MT bundles, the salt concentration is brought to
350 mM NaCl by addition of high-salt lysis buffer and incubation for 15 min.
To maximize extraction, the lysate is homogenized using a Dounce homogenizer
(Wheaton) and the resulting lysate is centrifuged for 45 min at 45,000
g
to pellet
cell debris and inclusion bodies (
Fig. 3.3
B). A lipid fraction is present at the top of the
supernatant. The nonlipid supernatant contains soluble proteins and is removed for
further purification.
His-tagged DCX is purified by affinity chromatography: 1 mL Ni-NTA resin
(Qiagen) per 1 L of culture is equilibrated with wash buffer, then added to the soluble
lysate, and incubated for 1 h on a rotating wheel at 4
C. The relatively high imid-
azole concentration (50 mM) reduces nonspecific binding. The resin is then depos-
ited on a small gravity column and the flow-through discarded (
Fig. 3.3
B). Two
successive 5-column volume washes help minimize the contaminants bound to
the resin. The His-DCX is eluted with 250 mM imidazole. This step both purifies
and concentrates the protein of interest, yielding typically 20-30 mg in 3-mL buffer,
starting from a 2-L culture. However, His-tagged degradation products cannot be
separated, and other contaminants may remain.
The purified 42 kDa His-DCX is loaded on a HiLoad Superdex 75 16/60 column
(GE Healthcare) and elutes with a peak at 53 mL, monitored by AKTA FPLC (GE
Healthcare) (
Fig. 3.3
C). This step purifies the protein and allows buffer exchange but
dilutes it
10-fold, typically yielding 8-12 mg protein in 10-mL buffer.
to extensive MT bundling. Scale bars
100 mm. (B) Extraction of DCX and initial
purification of His-DCX by affinity chromatography. SDS-PAGE and Coomassie staining.
MW, protein molecular weight marker (NEB); p and s, pellet and supernatant of cell
lysate centrifuged 45 min at 45,000g; FT, flow-through of first Ni affinity column;
W1, wash 50 mM imidazole; W2, second wash; E, elution 250 mM imidazole. (C) Gel
filtration profile of His-DCX monitored by UV absorbance and SDS-PAGE. (i) UV absorbance
read at the exit of an S75 16/60 size-exclusion column injected with 3 mL His-DCX
Ni-NTA elution, run at 0.5 mL/min. The 1 mL fractions analyzed by SDS-PAGE are
highlighted in yellow. The inset shows a gel filtration profile of cleaved DCX, with a bigger
aggregates peak (first peak). (ii) SDS-PAGE and Coomassie staining of fractions 6-18
and 27. Fractions 7-17 were pooled. (D) TEV cleavage of the His-tag. SDS-PAGE and
Coomassie staining. MW, protein marker; His-DCX, gel filtration pooled fraction;
¼
TEV,
same with addition of rTEV and overnight dialysis—observe the band shift; FT_Ni-NTA, flow-
through of the second Ni affinity column which retains uncleaved protein and His-tagged
rTEV.
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