Biology Reference
In-Depth Information
18.
Add EDTA and PMSF to a final concentration of 5 m
M
and 3 m
M
, respectively.
The following steps should be carried out in the cold (4
°
C) using prechilled
reagents and equipment.
19. Immediately dialyze against 20 L of 50 m
M
NH
4
HCO
3
pH 8.0, 5 m
M
EDTA,
1 m
M
PMSF with at least two changes of buffer (
see
Note 17
).
20.
C) to remove any cell
debris or precipitates that may remain. Load onto a 100-mL conventional
Heparin-Sepharose column. The unbound can either be discarded or passed
through the column again for optimal recovery of recombinant, so long as the
maximum column binding capacity has not been reached. Wash column with an
equal volume of the same buffer (
see
Note 18
).
Centrifuge the dialyzed medium at 16,500
g
for 30 min (4
°
21.
While following A
280
, elute bound material from the column with a 200-mL lin-
ear 0-1
M
NaCl gradient, collecting 150 drop fractions. If you are not equipped
with a conductivity measurement device to follow salt concentration, determine
the conductivity of every other fraction of eluant. Pool fractions from 18 mS to
the end of the A
280
peak. Alternatively, fractions can be analyzed by SDS-PAGE
and protein staining and pooled based upon the position of the recombinant. For
a typical elution profile,
see
Fig. 2A
.
22.
Dialyze pooled fractions versus 150 m
M
Tris HCl pH 7.4, 1 m
M
EDTA, 0.5 m
M
PMSF (
see
Note 19
).
23.
Load onto a conventional 25 mL DEAE-sepharose column equilibrated in the
same buffer, collecting the unbound protein (
Fig. 2B
).
24.
Centrifuge
unbound
protein at 230,000
g
for 20 min (4
°
C).
25.
Inject onto a Heparin 5PW FPLC column with a flow rate of 1 mL/min and elute
bound material with a programmed linear gradient of 0 - 1
M
NaCl in the same
buffer (
see
Note 20
). For a typical elution profile
see
Fig. 2C
(
see
Note 21
).
The rG peak elutes at conductivity of approx 32 mS. Pool fractions containing
the recombinant, concentrate with Aquacide and dialyze against TBS
50
, 0.02%
NaN
3
. Determine recombinant protein concentration, homogeneity, aliquot, snap
freeze in liquid nitrogen, and store at -120
°
C.
4. Notes
1.
Antibodies were most useful for immunoblotting rather than other immunologi-
cal techniques.
2.
The lepidopteran cell line used in our laboratory is Sf9, derived from the parental
cell line Sf21ovarian cells of
S. frugiperda
. We have compared baculovirus
expression in Sf9 and High Five cells (
Trichoplusia ni; Invitrogen
) and found no
significant differences for recombinant laminin expression. Sf9 cells grow at
27
C with ambient CO
2
and can be weaned from serum-dependence for growth.
Additionally, they can be reversibly maintained in culture as adherent monolay-
ers or suspension cultures. These growth conditions and characteristics are opti-
mal for recombinant protein expression and purification: the absence of serum in
growth medium reduces overall protein levels permitting easier purification of
the recombinant while still providing carrier protein; the ability to grow in
°
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