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The followmg procedure IS written for use with the Pharmacia FPLC or
GradlFrac systems.We use a 5-mL heparm column from Pharmacla. The column
1srun at room temperature. Buffer A IS PBS and Buffer B IS PBS/l .5M NaCl.
Gradient composltlon and times need to be optimized for each mdivldual protein
1 Equilibrate the column with Buffer A then wash the column with Buffer B until
the A 280nm IS constant (baseline)
2 Re-equilibrate the column with 5 column volumes of Buffer A
3 Load the filtered sample at 1 0 mL/min. We load 600 mL of supernatant contam-
mg 6 mg of gC-2(426t)
4. Wash with 5 column volumes of Buffer A
5. Elute the protein with a gradient from O-60% Buffer B over 75 mm (collect 1-mL
fractions). Next run a gradient from 60-100% B over 10 mm followed by a 5-mm
wash with Buffer B in order to ensure that all protein IS removed from the column
6 Wash the column with 5 column volumes of Buffer A
7 If this is the final column run, wash the column with 5 column volumes of 20%
EtOH Store the column at 4°C for future use
8. Pool the protein-containing fractions, dialyze against PBS, and run the pooled
sample over the column again using the same gradient Pool and concentrate the
protein-containing fractions and dialyze against an appropriate storage buffer
Store the protein at -80°C
147
Secreted HS V Glycopro terns
3.5.3. Gel Filtration Chromatography
Concentrated samples of immunoaffimty-purified gC and gD are yellowish
in color. This appears to be owing to residual components of the culture
medium that remain after the initial purification step. Gel filtration chromatog-
raphy successfully removes the remainmg traces of medium from the protem
sample. We routmely use a Pharmacia Superose 12 column (useful for proteins
in the 1 x 103-3 x lo5 mol-wt range).
1 Fully equilibrate the column with PBS not exceeding a flow rate of 1 mL/mm
Thts IS best done as an overnight wash.
2 For best resolution, the volume of the sampleshould not exceed2% of the total
bed volume of the column.
3 Filter the sample through a 0.2-pm filter and load the sample onto the column
The flowrate should not exceed 1 mL/min for the duration of the run
4 The running buffer for the entire experiment is filtered PBS.
5. Protem size will dictate how long after the void volume the protein will elute To
be safe, it IS best to start collectmg fractions a short time before the void volume
of the column has passed through
6. Collect OS- to I.O-mL fractions Analyze the fractions by SDS-PAGE and/or
isoelectric focusing (IEF) to determine which ones to pool Pool and concentrate
the approprtate fractions, dialyze against an appropriate storage buffer, ahquot,
and store the protein at -80°C
7 After all of the protein has eluted, wash the column with 5-10 bed volumes of PBS.
Clean and maintain the column according to the manufacturer™s specifications
8. Store the column in 20% EtOH
3.6. Characterization of Purified Protein
Below we provide a brief description of the methods we have used to char-
acterize our baculovuus-expressed proteins. Detatled protocols are not given,
and certainly more methods are available than the ones we describe.
3.6.1. SDS-PAGE and Western Blotting
SDS-PAGE is useful if it IS possible to purify the protein from the cell
supernatant or extract, or if enough of the protein is made so that is stands out
from the other baculovirus infected cell proteins present m your sample. Silver
staining 1smore sensitive (0.2-0.5 ug of protein/lane; we use the Pharmacia
Plustone Protein Silver Staining Kit) than Coomassie staining (l-l 0 yg of protein/
lane; we use the ISS Pro-Blue Staining System, Natick, MA). SDS-PAGE IS
used to get an approximate molecular weight and to determme the homogene-
ity of the sample.
A Western blot will indicate which of the stained bands contam the protein
(0.05-l pg of protein/lane). Western blot analysis is better than a simple
Willis et al.
148

tmmuno-dot blot because the different species present in the sample are sepa-
rated and can be distinguished from one another. A Western of “native” PAGE
(7) is useful to probe the antigenic conformation of the protein with MAbs that
bmd to discontinuous epitopes However, keep in mind that not all MAbs react
well in a Western blot of a denaturing or native gel.
3.6.2. Isoelectric Focusing
If the purified protein is going to be used for crystallography, IEF IS essen-
tial because proteins that are homogeneous on IEF tend to crystallize better
We have had successusing two IEF systems, the Pharmacia PhastSystem, and
the IEF gels that can be purchased from Novex. Both systems give essentially
the same results, however the Novex (San Diego, CA) gels are easier to
transfer for Western blot analysis. In general, 200-300 ng of protein are
needed per lane for a PhastSystem IEF gel and 0.5-l pg of protein are needed
per lane for a Novex IEF gel.
3.6.3. Quantitative ELISA
A quantitative enzyme-linked nnrnunoadsorbent assay(ELISA) more critically
assesses binding of MAbs. The assayis sensitive enough to determine differ-
the
ences in binding among MAbs that are not detectable on Western blots. In addi-
tion, some MAbs do not react well in Western blots even of native gels, and we
have found that theseMAbs react much better in the ELISA. We have also used the
ELISA to look at the effect of heating the protein on MAb binding (23).
Briefly, coat 8 pg/mL protein in PBS on 96-well microtiter plates (Corning,
Cornmg, NY) for 2 h at room temperature. Remove the remammg sample and
block nonspecific binding by adding 1% bovine serum albumin (BSA), 1% oval-
bumm in PBS. Remove the blockmg solution and wash the wells with 0.1%
Tween20/PBS. Serially dilute MAb ascites fluids in blocking solution and add
for 30 mm at room temperature. Remove the unbound MAbs and repeat the wash.
Next add protein A-horseradish peroxidase (Boehrmger Mannheim, Indianapo-
lis, IN) for 30 mm (remove and wash as above) followed by the substrate 2,2™-
azmo-di(3-ethylbenzthiozoline-6-sulfonrc acid [ABTS]). Allow the reaction to
proceed at room temperature until the optimal color change is achieved, approx
1.5 absorbance units in the darkest well. We assessabsorbance with a Dynatech
(Dynatech, Chantilly, VA) plate reader using a 405˜nm filter blanked against a
negative control well (31) Alternatively, twofold serial dilutions of protein can
be coated onto the plates and probed with a fixed dilution of MAb.
3.6.4. Analytical Gel Filtration
This technique is useful to get an approximate molecular weight of the pro-
tein. The molecular weight obtained using gel filtration can be compared to
Secreted HS V Glycopro terns 149

that obtamed usmg SDS-PAGE. Comparison of the molecular weights by the
two techniques will give a good indication if the protein exists as a monomer,
dimer, or higher-order structure in solution.
Calibrate the column usmg a combination of high and low molecular weight
standards (purchased from Pharmacia). We use a Superdex 75 column (HR 1O/
30, Pharmacla) for our analytical gel filtration work. Run a sample of the pro-
tein over the column using the same procedure used to run the protein stan-
dards (in general use a small volume, 100-200 uL at a protem concentration
0.5-I mg/mL). The molecular-weight standards come with dlrectrons for cal-
culating molecular weight based on the elution volume.
3.6.5. Physical Characterization
We routinely use a number of physical techniques to verify and characterize
the sequence, conformation, and oligomeric state of our recombinant proteins.
Brief descriptions of each are offered in the followmg.
3 6.5.1. N-TERMINAL SEQUENCING
It is often beneficial to confirm that correct N-terminal processmg has taken
place in the insect cells by submitting a sample of purified protem for N-termi-
nal sequence analysis. Sequencing takes very little protein (200 pmol or less),
and we have found that many of the preparations contain a small percentage of
protein (<IO%) that has been clipped at the N-termmus.
3.652. MASS SPECTROMETRY
This technique is usefil to get an accuratemolecular weight of the protem and to
help judge the purity of the sample.The amount of protein neededdepends on the
type of massspectrometrybeing done but should be m the IO-pm01range. We have
found matrix-assistedlaser desorpttonionization massspectrometty(MALDI) use-
ful in characterizingbaculovirus-expressedglycoproteins (I, 18™. Mass spectrometry
must be done m the absence detergents.
of Unless dimers and higher order structures
are stabilizedby covalent bonds, the molecular weight obtained will be of the mono-
mertc form of the protein becausedimers andhigher order structuresare disrupted by
the ionization procedure necessary to collect the mass spectrometry data. The
molecular weight obtained using this technique will be the most accurate because
anomalous effects owing to nngration through a column or gel do not have to be
considered.Comparing this value to the formula weight will also give a good mdica-
tion of the extent of posttranslationalmodification, e.g., glycosylation.
3.653. SCANNING TRANSMWON ELECTRON MICROSCOPY
STEM IS anothermethod of looking at the molecular weight of a protein m
solution and is also useful to determine the oligomeric state of the protein.
Willis et al.
150

Unlike the more traditional methods of molecular-weight determination that
measure averages over a total population (SDS-PAGE, mass spectrometry),
STEM quantitates the mass of one molecule at a time and also looks at the
molecule™s size, shape, and Internal mass distribution (33) Less than 30 pmol
of sample are needed for STEM analysis. A histogram of the data is made to
look at the overall mass distribution, and the average molecular weight is
determined. The average molecular weight can be compared to that obtamed
from mass spectrometry to determine the ohgomeric state of the protem.
3 6.5.4 CIRCULAR DICHROISM

CD measures the difference m a protein™s absorbance of right and left circu-
larly polarized light (5), and the CD signal m the far UV region is used to probe
the secondary structure of proteins. CD experiments can be used to estimate
the quantity of secondary structural elements m the proteins being studred and
to compare them one to another This technique must be carried out m aqueous
solution, preferably m dilute buffers (I 0). The concentration of sample needed
depends on the molecular weight of the protein and the path length of the cuvet.
We had successusing 0.3 mg/mL solutions (25 mMKP1 buffer pH 7.2) of gD-
l(306t) and gC-l(457t) m cuvets with pathlengths of 1 and 0.02 mm. CD has
been useful m comparing a panel of gD variants to determine if any of the
ammo acid changes have an effect on the secondary structure of the protein
3.6.6. Crystallography
X-ray diffractton of protein crystals is one of the ultimate ways of determm-
mg the structure of a protein. In general, vu-al glycoprotems have not been
frequently studied using this method because of the large quantity of protein
needed for successful experiments. The baculovuus expression system helps
bypassthis road block. For crystalhzatron,protem samplesneed to be highly homo-
geneous on SDS-PAGE, native PAGE, and IEF gels. The protein also needs to
be stable at high concentrations (10 mg/mL) and m low iomc strength buffers
For crystallization we use a two-step purrfication (immunoaffimty followed
by gel filtration chromatography). The protein is then concentrated and
exchanged mto 25 mM HEPES, pH 7 0. It is best to avoid phosphate buffer
because phosphate crystals are formed easily. The final concentration of pro-
tein should be 10 mg/mL. The best starting point for screenmg crystallization
reagents is to purchase a Crystal Screen Kit (Hampton Research, Riverside,
CA). The kits provrde a variety of crystallization solutions covering a wide
range of pH, precipitants, and salts that have been commonly used in the crys-
tallization of macromolecules. There are a number of crystallography books
that are good for the first time crystallographer to read (12,20,22,24).
151
Secreted HS V Glycopro terns

4. Notes
4.1. Which Cell Line and Which Medium to Use?
Our initial studies to determine the optimal insect cell lme and insect medium
were confusing. After much deliberation we settled on Sf9 cells grown in
serum-free SF-900 II medium. The Sl9 cells were weaned onto this medium by
the company and these adjusted cells can be obtained directly from Gtbco.
Other companies make then own formulation of serum-free medium and have
adapted insect cells to their formulation. We suggest you pick one cell line and
stay with it. Pluronic F-68 is Included m SF-900 II medium as an antishearing
agent so there is no need to supplement.
4.2. Cell Line Maintenance
New suspension cultures are started every 3 mo from frozen cells or if there
is a noticeable drop of either virus titer or protein production. Cells are thawed
and placed directly in suspension culture (27°C SF-900 II medium, no FBS)
and kept at 1 x lo6 cells/ml until they start to grow. The cells should be sub-
cultured every 3 to 4 d and should be ready to work with m 1wk. Thereafter the
cells can be maintained at 5 x lo5 cells/ml (see Section 3.2.2.). We recom-
mend using a 27°C refrigerated incubator for culturing cells and growing virus
to avoid the ups and downs of “room temperature.”
4.3. Cloning and Purification of Recombinant Virus
We have had excellent results using BaculoGold DNA (Pharmingen) for
doing homologous recombmation. Our efficiency for positive plaques ranges
from 60-l 00% (Fig. 3). We have not had successusing St9 cells directly from
a spinner flask for transfection. Monolayers should be split at least twice for
the best transfection results (see Section 3.2. I .).
When doing plaque purification, it is best to choose the plate with the lowest
density of plaques. Be sure that each picked plaque 1s derived from a single
recombinant virus, not a possible mixture of clones.
Assessing protein productton by Western blotting is better than looking by
Coomassie blue stammg becausethere are also baculovnus proteins m the me-
dium. It is also more informative and easier to screen directly for protein expres-
sion rather than probing for the gene. The high-salt concentration of the SF-900
II medium influences the migration of proteins on SDS-PAGE. The proteins ap-
pear larger (slower mobility) when electrophoresed in the presenceof medium
The 2% Seaplaque agarose used for titermg the vn-us can be prepared in
large quantities, autoclaved, ahquoted, and stored at room temperature. Micro-
wave aliquots carefully to reheat. Once overheated, the agarose loses its ability
to solidify.
152 Willis et al.

4.4. Storage of Virus
Virus stored at 4°C loses one full log of mfectlvlty over a 6-mo perrod. In
addition, the vu-us 1slight sensitive and should not be stored m a frequently
used cold room. For these reasons, we store all virus in convenient aliquots at
-80°C m SF-900 II medium supplemented with 5% FBS. There is no loss of
mfectlvlty on thawing, however, we do not refreeze the allquots. In addition,
once you know your clonmg has been successful, freeze all of your trans-
fectants and all of your plaque purified clones at -80°C m case you ever need
to go back to them.
4.5. High-Titered Virus
It is important to obtam high-titered virus (at least lo8 PFU/mL) for mfec-
tlon of large volumes of cells In addition, it 1scritical to titer the vu-us stock
carefully so that the mol can be carefully controlled. Fresh, healthy cells grow-
mg m suspension m log phase (95-l 00% vlablllty) are the key to getting a high
titer vu-us stock. To test for the proper moi, infect lOO-mL allquots of cells
at 2 x 106/mL with a range of recombinant vn-us from 0.1 to lPFU/cell follow-
mg the protocol m Section 3.3.5. We have found no evidence for the genera-
tion of defective viruses. Each virus stock is then tltered and the best moi
determined.
4.6. Viewing the Plaques
Viewing plaques 1sa major drawback of the baculovirus system. The best
method for viewing 1swith a dlssectmg scope and a stereo light source such as
Sage Instruments-Model 28 1 or a Leltz Wild MZ8 stereoscope. Excellent defi-
nition can also be obtained with side lighting usmg fiberoptics. The picture in
Fig. 2 gives an example of what the plaques look like.
4.7. Growing Cells in Suspension Cultures
Adequate aeration 1svery important for growing Sf9 cells m suspension and
can be achieved m any spinner culture of 500 mL or less using a stirrmg paddle.
The problem arises when volumes of greater than 500 mL are needed, Maxl-
mum aeration of the culture is absolutely mandatory particularly after mfec-
tlon. If aeration 1snot maintained at a high level we see no increase m protein
production from the zero time point, so although the cells look infected, pro-
tein is not being produced
For volumes over 500 mL a double-paddle spinner 1s necessary (Bellco).
The top paddle should be set to break the surface of the culture. We do not see
damage to Sf9 cells usmg these paddles. For larger spinners (3-8 L) we employ
an overhead drive (100 rpm) and a sparger to enhance aeration (control valve-
MG Industries, Philadelphia, PA). Foaming caused by bubbling the air mto the
153
Secreted HS V Glycoprotems
vessel does no harm. There 1sno reason to concentrate the cells prior to adding
virus to obtain efficient mfection and production of protein.

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