Biology Reference
In-Depth Information
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FIGURE 11.1
Purification of FIT2-His
6
-StrepII. Recombinant FIT2-His
6
-StrepII protein was purified,
subjected to electrophoresis on 15% SDS-PAGE, and visualized by Coomassie staining.
“Input” designates total cellular lysate; “Flow-through” designates protein not bound to
Strep-tactin resin; “Elution” designates protein bound specifically to column. Arrow
indicates purified FIT2-His
6
-StrepII.
Adapted from
Gross, Zhan, and Silver (2011)
.
11.2.1.6
Micellar detergent exchange performed on recombinant
purified proteins
Determining protein monodispersity is a critical prerequisite for crystallographic or
structure-function studies and demonstrate that the protein is not aggregated when
purified in the micellar compartment. If purified proteins were desired for gel
filtration chromatography in micelles other than Fos-choline 13 or Fos-cholines of
different acyl chain lengths, insect cell pellets were extracted in 1% Fos-choline
13 (w/v) overnight at 4
C, washed in Buffer C, and eluted in Buffer D as
described previously. However, after concentrating the protein to 1
m
g/
m
l in Buffer
F, buffer exchange was performed on Amicon Centrifugal Filter Units using
another detergent (i.e., dodecyl maltoside or
n
-octyl-
b
-
D
-glucoside) at a concentra-
tion that was four times its critical micelle concentration in Buffer A with 10%
glycerol (w/v). Monodispersity was assessed in a variety of detergents by gel
filtration chromatography and secondary structures in these detergents determined
by circular dichroism spectrophotometry (see below). Fos-choline detergents of
larger or smaller acyl chain length were assessed (Fos-choline 8, Fos-choline
10, Fos-choline 12, and Fos-choline 14), none of which displayed the same extent
of monodispersity as FIT2 in Fos-choline 13. Thus, Fos-choline 13 is an ideal
detergent to be used if FIT2 protein is to be crystallized in detergent micelles or
