Biology Reference
In-Depth Information
hand, DCX protein expressed in
Spodoptera frugiperda
insect cell cultures was stable
and could be readily studied in the variety of ways described below. Others have had
success subsequently with molecular studies of bacterially expressed DCX including
recapitulation of DCX's selectivity for 13-pf MTs (
Bechstedt &Brouhard, 2012
), but
the proteins' concentrations used were typically lower than required for our
structural studies.
3.2.1.2
Expression of DCX
The 366-amino-acid isoform of human DCX was cloned in the Invitrogen Bac-to-
Bac system by Dr Fiona Francis, Institut du Fer
`
Moulin, Paris. This isoform is a
splicing variant from the 360-amino-acid form, containing five residues (GNDQD)
inserted after residue 310 and one Val inserted after residue 342 (nomenclature of the
360-amino-acid form; NCBI Reference Sequence database); the significance of these
differences is not understood. The engineered construct includes in its N-terminus a
6-Histidine affinity tag (His-tag), a TEV (tobacco etch virus) protease cleavage site
(ENLYFQG, cleavage occurs between Q and G), and a Flag-tag (DYKDDDDK)
used originally for immunofluorescence in transfected cells (
Sapir et al., 2000
).
The baculovirus genome containing the tagged DCX gene was transfected into
Sf9 cells and amplified using standard methods (Bac-to-Bac Baculovirus Expression
System, Life Technologies). Test expressions of DCX constructs were performed in
small flasks; fewer cells with larger average diameters are indicative of baculovirus
infection. Sf9 cells are usually round but the expression of DCX constructs causes
their shape to change: they become lemon shaped or even grow a long cytoplasmic
extension, which is presumably due to extensive MT bundling by DCX (up to
200
m
m) (
Fig. 3.3
A).
For large-scale protein expression, cells were passaged to 450-mL cultures in 2.5-L
spinners containing fresh medium, to a final concentration of 1
10
6
cells/mL.
10
6
cells/mL at the infection
time point 24 h later. In our hands, 50 mL P2 was added to a 450-mL culture to
achieve a multiplicity of infection (MOI
They were grown for 24 h, so that they reached
2
number of virions/number of cells) of
3-10, which stops cell multiplication and induces protein expression. Cultures are
incubated for 48 h at 27
C. Cells are harvested by centrifuging in 1-L tubes at
700
ΒΌ
g
for 20 min at 4
C in a Beckman Avanti J-20 I centrifuge. Pelleted cells were
washed with 40 mL cold PBS per 1 L of culture and centrifuged in 50-mL tubes at
700
g
another 20 min in an Eppendorf 5810R centrifuge at 4
C. Pellets were frozen
in liquid nitrogen and stored at
80
C until protein extraction.
3.2.1.3
Purification of DCX
The protocol originally described in
Moores et al. (2004)
was modified slightly to
improve the solubility and minimize protein degradation, together leading to better
yields. All protein purification steps and centrifugations were performed at 4
Cto
limit protease activity. Buffers used are listed in
Table 3.1
.
Extraction of cytoplasmic proteins is performed by incubating frozen cell pellets
(typically 20 mL for 2-L cultures) with an equal volume of hypotonic lysis buffer
