Biology Reference
In-Depth Information
11.1
MATERIALS
11.1.1
Buffers
Buffer A contains 50 mM Tris-HCl pH 7.4 and 150 mM NaCl. Buffer B contains
Buffer A and 1 mM EDTA supplemented with EDTA-free protease inhibitor tablets
(Roche). Buffer C contains Buffer A and 0.1% (w/v) Fos-choline 13. Buffer D con-
tains Buffer C, 2.5 mM desthiobiotin, and 10% (w/v) glycerol. Buffer E contains
10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% (w/v) Triton
X-100. Buffer F contains Buffer C and 10% (w/v) glycerol.
11.1.2
Other materials
[9,10-
3
H(N)]-triolein ([
3
H]-TAG, 106 Ci/mmol) (Perkin Elmer), [1,2,3-
3
H]-
dioleoyl-
rac
-glycerol ([
3
H]-DAG, 30 Ci/mmol) (American Radiolabeled Chemi-
cals), all other lipids (Sigma Aldrich and Avanti Polar Lipids), Fos-choline 13
(
n
-tridecylphosphocholine) (Anatrace by Affymetrix), Triton X-100 and desthiobio-
tin (Sigma), pcDNA3.1-V5-His
6
vector, Lipofectamine 2000, Cellfectin II, insect
cell media (Invitrogen). The suppliers for all other materials are indicated in the text.
11.2
METHODS
In this section, we describe techniques for expressing recombinant integral mem-
brane proteins in insect cells and mammalian cells and purifying integral membrane
proteins in detergent micelles. We further elaborate on biochemical techniques for
determining protein-lipid-binding affinities and capacities and describe a protocol
for the generation of proteoliposomes for biochemical and reconstitution studies.
11.2.1
Purification of integral membrane proteins
11.2.1.1
Cell culture of HEK293 cells
A high-throughput assay can be utilized to screen for binding characteristics of a
variety of protein variants (i.e., a mutant library of a particular protein). To this effect,
we have developed a cell culture-based approach of expressing and purifying integral
membrane proteins and testing their lipid binding in detergent micelles.
To this end, monolayers of human embryonic kidney (HEK293) cells were
obtained from American Type Tissue Culture Collection and cultured in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal
bovine serum (FBS-HI) and 1
penicillin-streptomycin (DMEM-FBS) at 37
C
in a 5% CO
2
incubator. In order to purify 2-4
m
g of FIT2 (a protein that we used
to develop this technique), we utilized six-well tissue culture plates seeded with
1-1.5
10
6
HEK293 cells/well.
HEK293 cells reliably and reproducibly expressed the highest amounts of
FIT2 protein when transiently transfected with a mammalian expression vector
