Biology Reference
In-Depth Information
50
45
40
35
30
25
20
15
10
5
0
0
5
10
15
20
pmol [
3
H]-TAG added
FIGURE 11.7
The dose-response curve generated using cell culture-based assay. Saturable binding
kinetics using cell culture-based assays are similar to those using insect cell-purified
recombinant protein—compare to
Fig. 11.5
. Data are represented as mean
SD.
Adapted from
Gross et al. (2011)
.
11.2.3.1
Preparation of proteoliposomes
Purified FIT2 was put into proteoliposomes as follows: 1-palmitoyl-2-oleoyl-
phosphocholine (POPC, Avanti Polar Lipids) or a preferred mixture of lipids was dried
down using nitrogen gas and resuspended in 1 ml of 50 mMTris-HCl, pH7.4, 150 mM
NaCl, with glass beads (Sigma). The suspension was vortexed for 1 min and placed on
ice for 10 min, vortexed again, and transferred to an Eppendorf tube by pipetting with-
out disturbing the glass beads. The suspension of POPC was made into multilamellar
vesicles by freeze-thawing between liquid nitrogen and a 42
Cwater bath.Multilamel-
lar vesicles were extruded using a Lipo-So-Fast extruder and 400 nm membranes
according to the manufacturer's instructions (Avestin). Mixed micelles of phospho-
lipids in detergent were prepared from unilamellar vesicles by addition of 1% Fos-
choline 13 (w/v) in Buffer A to final concentration 0.1% Fos-choline 13 and allowed
to rotate for 1 h at room temperature. FIT2 protein in 0.1%Fos-choline 13 was added at
a ratio of 1:1000 to 1:200 protein/lipid and rotated for 1 h at room temperature until
membrane proteins and phospholipids in detergent were equilibrated in the micellar
compartment. Detergent was then removed using Bio-Beads (Bio-Rad) in three succes-
sive steps at 4
C: the first and second, for 1-2 h each, and the third, overnight. In this
manner, proteoliposomes are formed as the detergent concentration decreases to a crit-
ical level and proteins spontaneously associate with phospholipids (
Rigaud, Levy,
Mosser, & Lambert, 1998
). Proteoliposomes were then centrifuged at 3000
g
for
5 min and supernatants were transferred to a new tube and used for experiments.
11.2.3.2
Limited trypsin digestion of proteoliposomes
As a crude measure of reconstitution of protein structure found in native isolated ER
membranes, proteoliposomes containing FIT2 and POPC with or without other lipids
