<<

. 21
( 61 .)



>>

peptide and standard baculovlrus vectors ylelded barely detectable amounts of
protein either mtra- or extracellularly (27). We also studied a construct con-
taming no signal peptlde and found that significant amounts of gD-l(306t)
were synthesized intracellularly, although as expected, the protein lacked car-
bohydrate, falled to fold properly, and was not secreted. Therefore the
Autographa californaa nuclear polyhedrosis virus (baculovirus) transfer vector
pVT-Bat (32) was used to insert the gD gene mto baculovlrus. This vector IS
derived from pAC373 (30) and contains the polyhedrin promoter and sequences
that normally flank the polyhedrin gene. The signal peptlde sequence is for
honeybee melittm, and cloning sites adjacent to this sequence permit m-frame
fusion of the coding sequence for a foreign protein with that of the mehttin
signal peptide. Tessier et al. (32) previously showed that use of this particular
signal peptide slgmficantly enhanced expresslon and secretion of the papain
precursor (EC 3.4.22.2) from baculovirus-Infected Spodopterafrugiperda (Sf9)
cells. For gD-l(306t) we routmely obtam 20 mg of lmmunoaffinity-purified
protein per liter of infected Sf9 cell growth medium After one additional step
of purification, the glycoprotein IS free of contammatmg proteins and appears
to be >99% pure.
Willis et al.
134

Gene of interest

f
2 weeks if
all goes well


i
Transfection
\
b
4-5 days



6 days

About
2 months

3 days



Western blot

LIII- 1 day
/



Virus stock buildup
,
-
4 days/passage



Fig. 1. Construction of a recombinant baculovirus. Plasmids with the glycoprotein
gene of interest were used as PCR templates to generate DNA fragments containing
the glycoprotein gene. The 5™ primer was designed to begin at the first residue of the
protein after the signal peptide and to be in frame with the mellitin signal peptide. The
3™ primer was designed to truncate the glycoprotein just prior to the hydrophobic TMR.
In addition, the 3™ primer was designed to add histidine residues to the carboxyl-termi-
nal end of the glycoprotein. Each primer contained an appropriate restriction site
(27,31). The vector pVT-Bat and the PCR-amplified glycoprotein gene were each
digested with complementing restriction endonucleases. The gene was ligated into
pVT-bat using T4 DNA Ligase. The ligated plasmids were used to transform E. coli
Secreted US V Glycopro terns 135

3.1. Steps Needed to Construct the Baculovirus Recombinant
Figure 1 summartzes the steps involved to obtain the desrred recombmant
protein, from cloning the gene through purifyrng the protem. The detarls are
given in Sectrons 3.2.-3.5. Section 3.6. descrtbes techniques that can be used
to analyze the protem, but these by necessity of space are given m less detail.
Notes on the protocols are given in Section 4.
3.2. Maintaining Cell Lines
Crttrcal to all of the followmg steps IS a good cell lme. Followmg the next
two protocols will ensure that your cell lme remains healthy.
3.2 1. Mamtainmg Monolayers of Sf9 Cells
Note: Monolayers of cells are only used for transfection.
1 Seed 7 x lo6 St9 cells from a spinner mto a T75 flask wtth Grace™s medium
(Gtbco, Gatthersburg, MD) plus 10% fetal bovine serum (FBS, BtoWhttaker,
Walkersvtlle, MD), 1X antibtotic/antimycottc (Gibco), and 0.1% Pluromc F-68
(Glbco, see Notes for choice of cells and medium)
2. When the cells reach confluence, split them (step 3) using a 1 3 or 1.4 dilution
Do not splat the cells lower than 1:4 because St9 cells grow best at a htgh mocu-
lation density It will take 3-4 d for the cells to reach confluence Incubate St9
cells under humtd condttions at 27°C with normal au condtttons.
3 Splittmg St9 cells:
a Remove the old medium from the T75 flask
b Add a few milhhters of fresh medium and sterile glass beads (3 mm, Thomas Scten-
ttfic, Swedesboro, NJ) to strip the cells off the wall of the flask, approx 50
beads for a T75 flask.
c Gently rock the glass beads against cells to dtslodge them.
d Gently ptpet up and down to break up the cell clumps. Place the cells mto a
new flask at the approprtate dtlutton
3.2.2. Maintaining Suspension Cultures of Sf9 Cells
Log-phase St9 cells m suspension have a doubling time of 24 h.
1 We routinely keep stocks of St9 cells growmg m suspension m a spinner with
SF-900 II serum free medium (Gtbco) plus 1X anttbtottc/anttmycotic Thts

Fig. 1 (cont™d) XL-1 -Blue competent cells. Plasmtds from ampicillin-resistant colonies were
screened by restriction endonuclease analysts. Posmve plasmtds were recombined into
baculovn-us using Baculogold as the source of baculovirus DNA Plaques were ptcked and
amplified Culture supernatants from mfected cells were screened for protem production
by SDS-PAGE and Western blotting using polyclonal anttserum. Postttve baculovu-us
recombmants were subJected to two additional cycles of plaque punficatton before amphti-
cationof thevirus stock.The time requiredto carryout thesesteps indicated the right.
IS on
Willis et al
136
enables us to have the cells available for scaling up for large infections We do
not supplement this medium wrth FBS
2 All spmners should be placed on a magnetized platform set to spm at the maxl-
mum speed. We use a Bell Stir Multr Stn 4 Magnetic Stirrer (Bellco, Vineland,
NJ) The surface of this magnetic stm-er does not get hot with prolonged use
3 Splnting cells m suspensron. Cells should be split to a density of no less than 5 x
IO5 cells/mL We spht cells twrce a week to keep them m the best condmon
4 Cells can be grown to as high as 6 x lo6 cells/ml for moculatmg large volumes
of media for large-scale protein productton
5 Long-term storage Freeze log phase St9 cells from a spinner at 2 x lo7 cells/ml
m 95% FBS + 5% DMSO. Store m hqutd nitrogen
3.3. Cloning, Transfection, and Amplification
of Recombinant Baculovirus
3.3.7. Transfection
We will not detail the steps in polymerase chain reaction (PCR) clonmg, or the
ligatron of the PCR product into pVT-Bat (Fig. 1) because these are well described
elsewhere (27,31,32). When designing the PCR prtmers it ts tmportant to remem-
ber that the coding sequence minus the normal signal peptide must be m frame
with the mehttm signal pepttde m the vector, and that several extra ammo acids
may be added at the N-terminus of the protein as a result of cloning. The resulting
vector transforms well mto XL- 1 blue cells (Stratagene, La Jolla, CA). DNA yields
from maxi preparations are on the order of 1 mg of DNA from a 500˜mL culture
Both CsCl-purified DNA and column-purified DNA (Qtagen, Chatsworth, CA)
work well for transfection. It IS Important to seed Sf9 cells from suspenston mto
monolayers at least 1 wk ahead of the transfectlon expenment (Section 3.2 1.)
1 From a monolayer of Sf9 cells, seed 3-4 x lo6 cells mto a 60-mm tissue culture
plate with Grace™s medium supplemented with 10% FBS, 0.1% Pluromc F-68,
and 1X antrbrotrc/antrmycottc Let sn for 1l/z h
2 Prepare the DNA solution in a sterrle 1 5-mL mlcrofuge tube* 0.5 pg BaculoGold
(Pharmmgen, San Drego, CA) linear DNA; 2 pg recombinant plasmrd DNA; 1
mL Grace™s medium (not supplemented, no FBS), 20 pL Insectin (Invltrogen
lrposomes, San Diego, CA)
3. Vortex the DNA solution vtgorously. Incubate at room temperature for 15 mm
4. Remove the old medmm from the Insect cells and replace rt wtth 2 mL of fresh
Grace™s medium (not supplemented, no FBS), let stt for 10 mm at room temperature.
5. Remove the medium from the plate, but do not let the cells dry (work quickly)
6. Add the DNA solutron dropwzse to the cells
7. Rock the plate slowly at room temperature for 4 h usmg a side-to-side platform
shaker (Rocker Platform, Bellco).
8 Add 1 mL of Grace™s medmm supplemented with 10% FBS, 0 1% Pluromc F-68,
and 1X antibrotrc/anttmycotrc
Secreted HSV Glycoproteins 137




Fig. 2. Plaques of bat-gCl(457t) infected Sf9 cells. St9 cells were infected with
tenfold serial dilutions of a bat-gCl(457t) baculovirus stock (31). Plaques appeared
after 6 d of incubation at 27°C.


9. Incubate the plate at 27™C for 4-5 d.
10. Remove the supernatant from the plate and save it. This is your recombinant
virus stock to be used for plaque purification. Store at 4°C in a sterile tube.
3.3.2. Plaque Purification
Plaque purification is a time-consuming but essential step to ensure that
100% of your virus stock is producing the protein of interest. Three rounds of
plaque purification will take approx 3 mo to complete (Fig. 1).
1. Split St9 cells growing in suspension to 5 x lo5 cells/ml one day before the
actual experiment. The next day, seed 7 x lo6 cells into a lo-cm plate. Note:
Make sure to pipet up and down to break up any cell clumps. This is necessary to
distinguish between a cell clump and a virus plaque (Fig. 2) after 5-6 d of incu-
bation. Allow the plate to sit for at least 30 min at room temperature for the cells
to adhere.
2. Make lo-fold serial dilutions from the recombinant virus stock using SF-900
II medium.
3. Remove the medium from the plate.
4. Add 3 mL of diluted viral supernatant (loss, 10e6,and lo-˜) to each plate. For the
first round of plaque purification, also use the 10-i dilution to make sure the trans-
fection worked.
5. Let the plates sit for 1 h at 27°C for infection to occur. Rock the plates occasionally.
Willis et al.
138
6 Prepare 2% Seaplaque agarose (FMC) m water Autoclave and keep m a 55°C
water bath until ready for use. Bring Grace™s 2X medium (20% FBS, 2X anttbt-
otic/antimycotic, 0 2% Pluromc F-68 ) to 37°C.
7 After the 1 h infection, mix equal volumes of 2X Grace™s supplemented medmm
with the liquefied 2% agarose. The final concentratton wtll be 1% agarose m 1X
Grace™s supplemented medium.
8 Remove the medium containing the vu-us moculum from the plate. Overlay the
plate with 10 mL of 1% agarose/lX Grace™s supplemented medium by pouring
carefully from one side of the plate
9 Let the plate sit undisturbed at room temperature until the agarose is completely
hardened (about 30 mm).
10 Incubate the plates m a moist 27°C incubator for 5-6 d. After this trme the plates
can be wrapped m parafilm and stored at 4™C. Leaving the plates too long at 27°C
wrll make countmg plaques more difficult Ftgure 2 shows typical baculovuus
plaques observed using a dtssectmg microscope.
I1 Pick a few plaques using a sterile long Pasteur pipet (cotton plugged). Elute the
plaque by immersing the pipet tip and releasing the piece of agarose mto 500 pL
of supplemented Grace™s medium (or SF-900 II medium) and mcubatmg over-
night at 4°C. Vortex the solutton the next day to release virus from agarose Pick
8-l 0 plaques

3 3.3. Amplification of Virus
Cycling through the preceding steps (Fig. 1) yields a vu-us stock m a small
volume (500 pL), The next step IS to make enough virus and protein for screening.
1 Using SF-900 II medmm, seed 1 2 x lo6 St9 cells from a spinner mto each well
of a 6-well plate Incubate the plate for at least 30 mm at room temperature to
allow the cells to adhere
2 Remove the old medium and add 1 mL of SF-900 II medium with 50 pL of the
solutton containing the vu-us from a single plaque (see Section 3 3 2 , step 11)
3. Let the infection proceed at room temperature for 1 h Slowly rock the plates on
a platform rocker during the infection
4. Add another 1 mL of medium to each well
5 Incubate the cells for 3 d at 27°C and collect the culture supernatant m a sterile tube
6. Run 10-30 uL of the collected culture supernatant on sodium dodecyl sulfate-
polyacrylamide gel electrophorests (SDS-PAGE). Check for protein productton
by staining for protem or Western blotting (Ftg 3).
7 Store the rest of the culture supernatant from each well at 4°C m a sterile tube.
8. On the basis of the Western blot (Fig. 3), pick one or two positive wells and
process the virus through the plaque assay and amphfication cycles again. Repeat
this procedure one or two more times (Fig. 1) until 100% of the plaques are post-
ttve for protein production by SDS-PAGE staining or Western blot analysts
Choose one virus stock to establish the permanent virus recombinant lme and
freeze the others at -80°C as backups.
Secreted HS V Glycopro teins 139

123456789




Fig. 3. Western blot analysis of gC-l(457t). Following SDS-PAGE proteins were
transferred to nitrocellulose and probed with polyclonal anti-gC antibody R46 (13).
Lane 1, cytoplasmic extract prepared from HSV-1 infected BHK cells (26); lanes 2-9,
20 pL of culture supernatants from individual plaques that were picked, placed into
medium and used to infect Sf9 cells.

3.3.4. Baculovirus Stock Titration
It is important to titer the virus stocksso that multiplicities of infection (MOI)
are known rather than estimated for large scale virus and protein production.
1. Split Sf9 cells growing in suspension to 5 x lo5 cells/ml one day before the
actual experiment. The next day seed 3 x lo6 cells into 60-mm plates. Note:
Make sure to pipet up and down to break up any cell clumps. This is necessary to
distinguish between a cell clump and a virus plaque after 5-6 d of incubation
(Fig. 2). Incubate the plate for at least 30 min at room temperature to allow the
cells to adhere.
2. Make lo-fold serial dilutions ofthe recombinant virus stock into SF-900 II medium.
3. Remove the old medium from the plate.
4. Replace the medium with 1 mL of the lCr5, lO6, and 10e7dilutions of the recom-
binant virus stock.
5. Let the plates sit for 1 h at 27°C for infection to occur. Rock the plates occasionally.
6. Prepare 2% Seaplaque agarose in water. Autoclave and keep in a 55°C water bath
until ready for use. Bring Grace™s 2X medium plus 20% FBS to 37°C.
7. Aspirate the virus and overlay the plate with 4 mL of a 1: 1 mixture Grace™s 2X
(plus FBS) and 2% agarose (see Section 3.3.2). The final concentration will be
1% agarose in 1X Grace™s medium. Leave the plate untouched for 30 min.
8. Incubate the plates for 54 d at 27™C in a humidified incubator. After this time
the plates can be wrapped in parafilm and stored at 4°C. Leaving the plates too
long at 27°C will make counting plaques more difficult. Leaving the plates for
less time at 27°C will decrease the accuracy of the titration. Count the plaques
using a dissecting microscope (Fig. 2) and calculate the titer. It is easiest to count
the plaques from the bottom of the plate.
Wills et al.
140

<<

. 21
( 61 .)



>>