Biology Reference
In-Depth Information
10
6
Sf9
cells on 60-mm Petri dishes and allow to attach for at least 30 min. Prepare 3 mL
serial dilutions (10
-4
, 10
-5
, 10
-6
, 10
-7
) of the recombinant viruses in TNM-FH
supplemented with 10% FBS. Remove the medium from the 60-mm plates and
add 1 mL of the virus dilutions on duplicate plates. Incubate the plates at 27
4.
Plaque-purify the amplified recombinant viruses (
see
Note 3
). Seed 2
×
°
C
for 1 h. Melt 3% SeaPlaque agarose in a microwave oven, cool to 37
°
C, and add
two volumes of the culture medium preheated to 37
C to obtain a 1% agarose
overlay. Remove the virus dilutions and add 4 mL of the agarose overlay. Incu-
bate the plates in a humid environment at 27
°
C for 6 d. Remove agar plugs con-
taining single clear plaques with a sterile Pasteur pipet (
see
Note 4
) into 1 mL of
the culture medium and elute the virus particles by incubation at 4
°
°
C overnight.
5.
Amplify the plaque-purified viruses three times for 3 d at 27
C (amplifications I,
II, and III) to obtain high-titer virus stocks (
see
Note 5
). After the first amplifica-
tion, perform test infections to screen the viruses for the production of the
recombinant proteins of interest (
see
Subheading 3.1.3.
). AI (amplification I):
seed 1
°
10
6
Sf9 cells on the wells of a 6-well plate and use 200
×
µ
L of the eluted
10
6
Sf9 cells on a 100-mm Petri
plaque-purified virus for infection, AII: seed 6
×
10
6
Sf9 cells on
dish and infect with 100
µ
L of the AI virus, and AIII: seed 6
×
a 100-mm Petri dish and infect with 5-10
L of the AII virus. The titer of the
AIII virus should be about 10
8
PFU (plaque forming units/mL).
µ
3.1.3. Expression of Recombinant Human Type III Procollagen
in Insect Cells
10
6
H5 cells (
see
Note 6
) on a 100-mm Petri dish and infect with the
rhproCIII and 4PH
1.
Seed 6
×
viruses. Use the former in a 5 to 10-fold excess over the
latter. Incubate plates at 27
αβ
°
C and add L-ascorbic acid phosphate (80
µ
g/mL) to
the culture medium daily.
2.
Harvest cells (
see
Note 7
) 72 h after infection, wash with PBS and homogenize in
the homogenization buffer (500
10
6
cells). Centrifuge the cell homo-
genates at 10,000
g
for 20 min and collect the soluble fractions. Analyze samples
with SDS-PAGE under reducing conditions, followed by staining with Coomassie
brilliant blue or Western blotting using polyclonal antibodies to the
µ
L/6
×
α
- and
β
-subunits of human prolyl 4-hydroxylase
(16)
, the N-propeptide of human type
III procollagen (Farmos Diagnostica, Turku, Finland) or a monoclonal antibody
95D1A recognizing the collagenous region of various collagen chains (A. Snellman
and T. Pihlajaniemi, unpublished observations). Estimate the amount of recom-
binant human type III procollagen by means of a PIIINP radioimmunoassay for
the trimeric N-propeptide of human type III procollagen, and assay the amount of
prolyl 4-hydroxylase activity by a method based on the hydroxylation-coupled
decarboxylation of 2-oxo[1-
14
C]glutarate
(17)
.
3.
The triple-helical conformation of the recombinant human type III procollagen
can be examined by pepsin digestion
(18)
. Lower the pH of the samples to 2.5,
add pepsin (1.5 mg/mL in 10 m
M
acetic acid) to a final concentration of 0.2 mg/mL,
and digest the samples for 1-4 h at 22
°
C. Inactive pepsin by adjusting the pH
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