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3.3 5 Growing Baculovirus Stock
As a general rule, vu-us stocks should be prepared at a low MO1 (0.1-0.3
PFUicell). We have occasionally found that some recombmants do not amplify
well unless the moi is at least 1. As with any vu-us, it is best to make large
amounts of low passagestock vnus before going to large-scale protein produc-
tion Our normal procedure is to save the first two passages of recombinant
virus stocks for growmg more vnus stocks in the future. We then use later
passagesfor protein production.
1 Bring at least 100 mL of St9 cells m SF-900 II medium to a density of 2 x lo6
cells/ml in a spmner
2 Infect cells at an mot of 0. l-l (depending on the recombinant vnus). At the same
trme, add 5% FBS to the medium Thts is the only time FBS IS added to SF-900 II
medium
3 Incubate the cells at 27°C for 96 h m a spmner flask Monitor the cell vtabtlrty
every 24 h by removing 1 mL of the culture, dtlutmg the cells l/IO with trypan
blue (a 0.2% solutton m phosphate-buffered salme (PBS) filtered through a 0 22-mm
membrane, Gtbco), and countmg the cells to determine cell denstty and vtabtlity.
It 1sImportant to wart to harvest the vnus until the cell vrabthty drops to 5&60%.
This usually occurs by 96 h postmfectton (PI)
4 Remove the culture from the spmner and pellet the cells by centrrfugatton
5 Save the supernatant as the vtrus stock.
6 Titer the vnus stock (see Sectton 3 3 4 )
7 Ahquot the vtrus stock and store at -80°C
3.4. Large-Scale Protein Production
In order to obtain multimilligram quantities of protein it is necessary to scale
up protein production to greater than 1 L. We routmely do 3-8 L preparations
using either a large Bellco spinner or a Celligen Plus Bioreactor (New
Brunswick Scientific, Hatfield, UK). Protocols for both are outlined m the fol-
lowing. Protein production can also be done in a shaker flask. Spinners are
easier to work with, however. Proper aeration IS very important for maximum
protein production. See Sectton 4.7 for a discussion of thts topic. Note that all
protein production is done m serum-free medium.
Samples are taken every 24 h for each run (described m the followmg) m
order to determine percent of cell vtability and so that Western blots can be
done to evaluate each run and compare one run to another. Figure 4 shows a
typical plot of cell density and percent cell viability vs time for cells infected
with bat-gDl(306t) (27). Also shown is a typical time course of protein pro-
duction by Western blot analysis. Do not be concerned rf there is protein in
your 0 h time-point. This is protein that is present m the recombinant vnus
stock used for inoculation.
Secreted HS V Glycopro teins

A B



12345




. - . - . - . - x-
0 20 40 60 a0 100
Time(h)

Fig. 4. Evaluation of protein production and cell viability of St9 cells infected with
bat-gDl(306t). Three L of St9 cells were infected with bat-gDl(306t) (27) at a moi of
4. Samples were taken daily and the cell density and viability evaluated. (A) Western
blot analysis of daily samples of gD-l(306t) from the culture supernatant (20 pL) us-
ing polyclonal anti-gD antibody R7 (1.5). Lane 1,0 h PI; lane 2, 24 h PI; lane 3,48 h
PI; lane 4,72 h PI; lane 5,96 h PI. (B) Plots ofpercent cell viability and cell density vs
time at 0,24,48, 72, and 96 h PI.

Spinner Cultures
3.4.1.
1. Bring an assembled and autoclaved Bellco spinner flask (3 or 8 L with double
paddle and sparger mechanism) into a sterile hood and add cells and medium to
the final desired volume and a cell density of 1 x 1O6 cells/ml.
2. Place the spinner in a 27™C incubator and attach an overhead motor. Spin at the
maximum speed.
3. Hook the sparging apparatus (same apparatus as used in fish tanks) up to a gas
tank (40% oxygen/60% nitrogen), and bubble air through the sparger at 5 cclmin.
4. When the cells have reached a density of 4.0 x lo6 cells/ml (l-3 d depending on
the initial cell density), infect with 4 PFU/cell of recombinant baculovirus.
Because recombinant virus stocks are usually lo8 PFU/mL, 100-200 mL of virus
stock will be added for a 3-L culture.
5. Remove an aliquot from the sample line (5-10 mL). Flush the sample line with
air from a syringe. Dilute 100 pL of the cell sample l/l0 with 0.2% trypan blue
(see Section 3.3.5.) and determine the cell density and percent cell viability. The cell
viability should be 95-l 00%. Spin the cells out of 1 mL of the sample. Freeze the
supernatant and save it as the 0 h time-point.
6. Every 16-24 h remove a sample as in step 5, count the cells and determine
the viability. Spin down 1 mL of the sample and save the supernatant as a
time-point.
Willis et al.
142

7 The cell density should remain consistent, although tt may increase during the
first 24-48 h and then level off
8. Harvest the culture when the cell viability drops to 70% (Fig 4) This occurs
between 72 and 96 h PI
3.4.2. Celligen Bioreactor
We have experience using the New Brunswick Celhgen Plus Bioreactor with
either a 5 L vessel (3.5 L working volume) or a 7-L vessel (5 L workmg vol-
ume) A detailed protocol is supplied by the manufacturer and will not be pro-
vided here. We can be contacted for a step-by-step walk-through of how we
use our Celhgen Plus Bioreactor.
New Brunswick sells two interchangeable systemsfor stirrmg the msect cell
culture: a marme blade and a cell lift. We have used both successfully but
recommend the marme blade because it is less expensive and sigmficantly
easrer to assemble. Infected cells m the Celhgen Plus Bioreactor produce up to
three times the amount of extracellular protein compared to similar runs made
usmg a large Bellco spinner. In addition, the length of a run m the Bioreactor is
at least 1 d shorter than a comparable run in a spinner. This may be owing to
the fact that both pH and O2 levels are electronically monitored and controlled
by the Bioreactor Normally, St9 cells at 4 x 1O6cells/ml are infected at a moi
of 4 Runs are momtored as described for the large spinner cultures (see Sec-
tion 3.4.1 ) Cell vtabtlity generally drops to 70% by 48-52 h PI
Settings for the Celhgen Bioreactor should be: O2 at 50%, pH = 6 2, tem-
perature at 27°C (although the machme should be calibrated at 25”C), agitation
set at approximately 140-200 rpm (opttmized for the stzeof the culture), mmal
cell density is 1 x IO6 cells/ml.
3.4.3 Harvesting Large Cultures of Infected Cells
It is easiest to harvest large runs by removing the cells by centrifugation.
1. Large spinner runs Remove the overhead drive and disconnect the bubbling
apparatus Pour the culture mto 1OO- to 150-mL centrifuge bottles and spm (4”C,
1400g) for 30 min If the protein is secreted save the supernatant, if not save the
cell pellet.
2. Celhgen btoreactor: Turn off the agitation and remove the culture vta the harvest
line according to manufacturer™s speclficattons Pellet the cells as described m
step 1
3. Samples can be stored at -20°C until ready to process further
3.4.4. Concentratron and Buffer Exchange
The Millipore Easy-Flow Masterflex tangential flow unit is convenient to
use and saves time when working with large culture volumes. The unit can be
143
Secreted US V Glycoproteins
used with a 0.4%pm cutoff filter to remove cells from the culture supernatant.
However, we find it more convement to remove the cells by centrlfugatlon (see
Section 3.4.3.). We routinely use tangential flow to reduce culture supernatant
volumes from 3,5, or 7 L runs to 1 L or less. It also is beneficial to use the unit
to exchange (dialyze) the sample with PBS. Exchanging the medium and salts
is a necessary step for some lmmunoadsorbent columns, possibly owmg to
inhibition of MAb binding. In general, the concentration step reduces column-
loading times and the buffer exchange increases the efficiency of chromatog-
raphy. However, this process is not beneficial for all glycoprotems, and for
gG2(426t) we found it to be detrimental. A detatled protocol for settmg up the
apparatus is provided by the manufacturer.
1, Assemblethe unit according to the manufacturer™s drrectlonswith the appropn-
ate membrane(we usea 10,000mol-wt-cutoff Pellicon Cassette Filter [Mllllpore,
Bedford, MA])
2. Flushthemembrane 10L of dH,O to removethe storingsolution(O.lNNaOH)
with
3. Sample preparation. Spm the sampleat 4°C and 10,OOOg 20 mm Filter the
for
samplethrough a 0 45pm disposablefilter (with a prefilter)
4. Cycle the culture sample through the membrane. We recycle the retentate back
mto the sample and discard permeate. The first time a sample is run through the
unit, however, save the permeate to ensure that the sample IS not flowing through
the membrane Continue until the volume has been decreased to approx 500 mL.
It IS convenient to use a 1-L graduated cylmder to hold the sample being concen-
trated so that the exact volume can be monitored.
5 Add PBS to the sample in the 1-L graduated cylmder. We routmely exchange
with a total of 5 L of PBS.
6. After all of the PBS has been added, bring the volume to 500 mL or less. Flush
the system with an additional 500 mL of PBS to remove any of the sample that
remains in the filter. The sample IS now ready for chromatography.

3.5. Protein Purification
The next step in the process is to isolate the protein from the medium. This
is most easily accomplished by using some form of affinity chromatography.
Two types of affimty chromatography successfully used tn our lab for large-
scale purification are described in the followmg: immunoaffimty chromatog-
raphy and heparin chromatography. Hlstidine-tailed proteins can be purified
on a small scale by N?+ -agarose chromatography (see Section 4.10.). Addi-
tional purlficatlon steps depend on the ultimate use of the protein. We next
purify our proteins over a gel filtration column. In addition, we have used amon
exchange to further purify our proteins, but have found that this third step of
purification is not necessary for the procedures we use. Keep in mmd that with
every purification step there IS a loss of protein. All protein purifications should
144 Willis et al.




e-200
4-116




Fig. 5. SDS-PAGE analysis of gD and gC variants expressed using a baculovirus
expression system. All proteins are truncated prior to the TMR. Purified glycoproteins
(l-5 pg/lane) were electrophoresed on 10% denaturing gels and stained with
Coomassie blue. (A) gC-l(457t) is a form of HSV-1 gC truncated at amino acid 457
(32). gC-l(A33-123t) is a form of HSV-1 gC lacking residues 33-123 in the N-termi-
nus and truncated at amino acid 457 (31). Both proteins were immunoaffinity purified
using MAb lC8 (13,31). gC-2(426t) is a form of HSV-1 gC truncated at amino acid
426 (31). The protein was purified by heparin chromatography (31). (B) gD-l(306t) is
a form of HSV- 1 gD truncated at amino acid 306 (27). gD-l(QAAt) is a form of HSV- 1
gD with mutations that remove the signal for the addition of the three N-linked oli-
gosaccharides (27,281. gD-l(QAAt) is also truncated at amino acid 306 (27). Both
proteins were immunoaffinity purified using MAb DL6 (13,27).


be monitored at A2s0 This can be accomplished two ways: using an automated
,,,,,.
system (Pharmacia [Uppsala, Sweden] Fast Protein Liquid Chromatography
[FPLC] or GradiFrac systems)that produces a chromatogram ofA2sOnm time, or
vs
measurmg A2s0 for collected fractions from a nonautomated column run.
nm
3.5.1. lmmunoaffinity Column
Immunoaffinity chromatography is the most convenient way to isolate gD
and gC from the infected cell culture supernatants (Fig. 5). We use CNBr-
Activated Sepharose 4B (Pharmacia, Uppsala, Sweden) to attach the purified
IgG (8 mg of IgG/mL of Sepharose) to the column matrix. Details on MAb
coupling are according to manufacturer™s specifications.
Secreted HS V Glycoproteins 145

1 2 3




Fig. 6. Western blot analysis of gD-l(306t). The protein was purified by immuno-
affinity chromatography on a DL6-sepharose column (13.27). Samples were electro-
phoresed on a 10% SDS-PAGE, transferred to nitrocellulose and probed with
polyclonal anti-gD antibody R7. Lane 1,20 pL of culture supernatant from SF9 cells
infected with bat-gDl(306t) for 96 h; lane 2,20 uL of column flow-through; lane 3,
1.2 ug of immunoaffinity-purified gD-l(306t).


Protein can be eluted from the column with either a high or low pH wash or
with a chaotropic agent such as 3M KSCN. The choice of eluant depends on
the protein and MAb being used. We use 0.1 M ethanolamine for high pH elu-
tion (Fig. 6). Our general running buffer for the column is TS wash (10 mM
Tris-HCl, pH 7.2, OSM NaCl).
All procedures are done at 4°C using a jacketed column (XK series,
Pharmacia), and all buffers are kept at 4°C in a water bath. If the immuno-
adsorbent column has been used five times or if the proteins being puri-
fied are switched, the column must be cleaned and regenerated according
to the manufacturer™s specifications. The regeneration protocol is
designed to remove proteins and other material that are bound non-
specifically to the column.
Samples should be run across the immunoadsorbent column according to
the following protocol:
1. Equilibrate the column at 2 mL/min with at least five bed volumes of TS wash.
2. Filter the sample through a 0.45pm filter before loading.
3. If you are using an automated system (i.e., Pharmacia GradiFrac or FPLC) write
a program with the following steps:
a. Load the sample onto the column overnight at a flow rate that does not exceed
2.5 mL/min.
b. Wash the column with five bed volumes of buffer A (TS wash).
c. Switch to buffer B (O.lM ethanolamine) in 2 min and run buffer B for 2
column volumes.
Willis et al

d Collect the eluted protein m an Erlenmeyer flask (on ice) and add 2 5M Tns-
Cl until the pH IS approx 7.0 Fifty mllhmeters of eluted protem needs approx
10 mL of 2 5M Tns-Cl to be neutralized
e. Switch back to buffer A m two minutes and wash the column with 5-10
bed volumes
4. Concentrate the protem to 1-5 mg/mL usmg the appropriate molecular weight
cutoff membrane We use the Amlcon 50 mL stirred-cell concentrator with YM 10,
YM3, or PM10 membranes The choice of membrane depends on protein size
However, some proteins unexpectedly go through membranes that should retam
them It 1swise to check the flow-through the first time a protein 1sconcentrated
5 Dialyze the sample against an appropriate storage buffer
6 Determine the concentration of the protem and store ahquots at -80°C

3.5.2. Heparin Chromatography
This procedure is used for the purification of gC-2(426t) because we have
not found an MAb suitable for the purification of this glycoprotem (all of them
irreversibly bind the protein), and we found that heparm chromatography is
better than Nl*+-agarose chromatography for large-scale purification. The
insect medium IS not exchanged with PBS, rather it IS run directly over the
heparm-sepharose column without concentration. By trial and error we found
this to be best for this protein. Two consecutive runs on the heparin column are
needed to purify gC-2(426t) (Fig. 5A).

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