Biology Reference
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if structure-function studies are to be undertaken. Novel proteins must be purified in
a variety of detergents in order to determine the ideal micellar lipid for biochemical
and biophysical studies.
11.2.1.7
Biophysical characterization of purified proteins
11.2.1.7.1
Gel filtration chromatography of purified proteins
Prior to performing lipid-binding assays, we performed gel filtration chromatogra-
phy in order to assess monodispersity of FIT proteins in a variety of detergents. This
is an important biochemical attribute that is critical if a protein is to be utilized to
study ligand binding or for structural studies. After calibration of a Superdex 200
HR 10/30 column with Thyroglobulin (670 kDa),
g
-globulin (158 kDa), ovalbulmin
(44 kDa), myoglobin (17 kDa), and Vitamin B12 (1.35 kDa), 100
m
g of purified
recombinant protein at a concentration of 1 mg/ml was chromatographed on the
column at a 0.5 ml/min flow rate in Buffer F. Elution of protein was detected at an
absorbance of 280 nm and FIT2 eluted at a MW corresponding to
60 kDa (Fos-
choline 13 micellar MW was
30 kDa)
(
Fig. 11.2
). In order to be able to perform gel filtration chromatography, purification
of recombinant protein from insect cells is required due to the large amount of protein
that is needed for this assay—cell culture yields are far too low.
30 kDa and recombinant protein MW is
kDa
670
158
44
17
20
15
10
5
0
0
5
10
15
20
25
Elution volume (ml)
FIGURE 11.2
Purified FIT2-His
6
-StrepII exhibits monodispersity on gel filtration chromatography. Using a
Superdex 200 HR 10/30 column, 100 mg of purified recombinant FIT2-His
6
-StrepII at a
concentration of 1 mg/ml was chromatographed in Buffer F and protein was detected at an
absorbance of 280 nm. The apparent molecular mass of FIT2-His
6
-StrepII is 60 kDa.
Standards are indicated with arrows: thyroglobulin (670 kDa), g-globulin (158 kDa),
ovalbulmin (44 kDa), myoglobin (17 kDa), and Vitamin B12 (1.35 kDa). FPLC traces are
representative of three independent experiments.
Adapted from
Gross et al. (2011)
.