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4.8. Protein Production
It is difficult to estimate the yield of protein secreted when working with
insect medium. The ionic strength of the medium and the presence of other
baculovnus proteins interfere with the protein assay. Accurate protein values
are obtained only after the glycoproteins are purified from the medium. We
have not found it necessary to use protease mhibitors when isolating proteins
from culture supernatants. We routinely obtain 5-30 mg/L of affimty-puriried
glycoprotein using our baculovirus expression system. In general, we try not to
refreeze purified protein so choose aliquot size carefully.
4.9. Immunoaffinity Chromatography
The scale-up from a small to a large immunoadsorbent is relatively straight-
forward. The total binding capacity of an individual IgG can be determined
only by trtal and error. For gD-l(306t) we use a 150-mL (8 mg of IgG/mL
of sepharose) column of DL6 IgG bound to CNBr-activated Sepharose
(Pharmacla, all procedures are accordmg to the manufacturer™s mstructions).
This column is able to bmd approx 90 mg of gD. The eluant for this column is
O.lM ethanolamme. It is very important to use fresh, colorless ethanolamme.
Old ethanolamme stock or old bottles of deteriorated ethanolamme should be
discarded. Old ethanolamme will strip antibody from the column. We now buy
ethanolamine in lOO-mL quantities from Sigma. Our immunoadsorbent col-
umns are remarkably stable and have been used repeatedly for several years
with no loss of activity. Between runs the columns are stored m TS wash/
0.02% azide at 4OC. Do not let the columns dry out.
4.10. Ni2™-Agarose Chromatography
We mitially used Nr*+-agarose(Qiagen) to purify small amounts of our proteins
from the infected cell culture supematantto take advantage of the histidine tail that
was added to the gC and gD constructs (27,31). When we tried to scale up the
purification, we encountered a number of problems. First, we were not able to load
multimilligram amounts of protein directly from the culture supematant onto the
column. Initially the protein bound, but adsorption dropped with time. Second, the
contents of the medium eventually stripped the Ni*+ from the column (changing
the color of the matrix from pale blue to white). Third, the pH of the growth me-
dium drops from 6.3-5.9 by 72 h PI (note that the pH is controlled m the Celhgen
Bioreactor) and mhibits binding. Finally, the Ni*+-agarosebeads are easily crushed
by extended spins in the micromge and under the low pressure used for FPLC.
Therefore, scale-up to a larger automated column was abandoned.
Willis et al.
154

We tried two different elution schemes for small-scale Nt*+-agarose chro-
matography. Both are protocols obtained from Qtagen. The first was to elute
the protein with a low pH wash and the second was to elute the protein with an
imtdazole wash (10 mA4 imidazole wash to remove nonspectfic binding fol-
lowed by a 250 mM imidazole wash to elute the protein). In general, protein
purified with imidazole elution looks cleaner on SDS-PAGE than protein puri-
fied by pH elution. Both procedures need to be optimized for the particular
protein being worked with.
4.11. Gel Filtration Chromatography
we use a UV monitor, we find It convenient to analyze the column
Although
fractions using the Pharmacia PhastSystem because small volumes (1 pL) and
protein amounts (I 50-200 ng) can be detected rapidly by silver stain. How-
ever, Western blotting or standard SDS-PAGE can also be used.

Acknowledgments
This work was supported by a Research Foundation Grant from the University
of Pennsylvania, Public Health Service grants AI-l 8289 from the National Instt-
tute of Allergy and Infecttous Diseases, NS-30606 from the National Instttute of
Neurological Disease and Stroke, DE-08239 from the Nattonal Instttute of
Dental Research, and HL-28220 from the National Heart, Lung, and Blood
Institute, and by a grant to G. H. C. and R. J. E. from the American Cyanamid
Co. S. H. W. is a postdoctoral fellow supported by Public Health Service grant
AI07324-05 from the National Institute of Allergy and Infectious Diseases. A.
V. N. is a predoctoral trainee supported by Public Health Service Grant NS-
07 180 from the National Institute of Neurologtcal Diseases and Stroke. A. H.
R is a postdoctoral fellow supported by Public Health Service grants AI07324-05
and AI07278- 10 from the Nattonal Instttute of Allergy and Infectious Diseases.

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2 Bmnetti, C. R., Burke, R. L., Kornfeld, S., Gregory, W., Mastarzt, F. R., Dingwell,
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6 Chiang, H -Y., Cohen, G. H , and Elsenberg, R J (1994) Identification of func-
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SIScoupled with Western blotting. J Vzrol 60, 157-166
8 Cohen, G H., Muggendge, M I., Long, D., Sodora, D. A., and Elsenberg, R. J
(1992) Structural and Functional Studies of Herpes Simplex Virus Glycoprotem D ,
in Genetzcally Engzneered Vaccines (Clardi, J. E , ed), Plenum, NY, pp 217-228.
9 Corey, L. and Spear, P G (1986) Infections with herpes simplex viruses N Engl
J. Med314,686-691, 749-757
10 Crelghton, T E (1989) Protem Structure, a Practical Approach, IRL, New York
11 Ducrmx, A. and Glege, R. (1992) Crystalllzatlon of Nucleic Acrds and Protews,
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12 Eisenberg, R J., Long, D., Sodora, D. L., Chiang, H. Y , Wilcox, W. C., Abrams,
W R., Muggendge, M I , and Cohen, G H. (1994) Structure and function of
glycoprotein D of herpes simplex w-us, m Frontzers zn Vzrology (Becker, Y and
Daral, G., ed ), Springer Verlag, Heidelberg, Germany, pp 43-65.
13. Eisenberg, R J., Ponce de Leon, M , Friedman, H. M., Fries, L F., Frank, M. M ,
Hastings, J. C , and Cohen, G H (1987) Complement component C3b binds
directly to purified glycoprotem C of herpes simplex vu-us types 1 and 2 Mzcrob
Pathog 3,423-435
14. Feenstra, V., Hodale, M , and Johnson, D. C (1990) Deletions m herpes simplex virus
glycoprotem D define nonessential and essential domains. J Vzrol 64,2096-2102
15 Isola, V. J., Elsenberg, R. J., Siebert, G R., Heilman, C J , Wilcox, W. C., and
Cohen, G. H. (1989) Fine mapping of antlgemc site II of herpes simplex virus
glycoprotem D J Vwol 63,2325-2334.
16. Johnson, D C., Burke, R L., and Gregory, T. (I 990) Soluble forms of herpes
simplex virus glycoprotein D bind to a limited number of cell surface recep-
tors and mhlblt vxus entry into cells. J. Vwol 64,2569-2576.
17. Johnson, D. C. and Llgas, M. W. (1988) Herpes simplex viruses lacking gly-
coprotem D are unable to Inhibit virus penetration* quantitative evidence for
virus-specific cell surface receptors. J Vu-01 62,4605-4612.
18. Karas, M. and Bahr, U (1993) Laser desorptlon ionization mass spectrometry of
bloorgamc molecules, m Methods in Molecular Bzologv Spectroscopic Methods
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and Analyses Mass Spectrometry, and Metalloprotern
Mulloy, B , Thomas, A H., ed.), Humana, Totowa, NJ.
19. Long, D , Wilcox, W C., Abrams, W. R., Cohen, G. H., and Eisenberg, R. J.
(1992) Disulfide bond structure of glycoprotein D of herpes simplex virus types 1
and 2. J. Vwol. 66,6668-6685.
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20. McRee, D E (1993) Practlcalprotein Academic, San Diego
crystallography,
2 1. Mtchel, H (199 1) Crystallizatzon of Membrane Proteuzs, CRC, Boca Raton, FL
22 Muggeridge, M. I , Roberts, S R , Isola, V J., Cohen, G H , and Eisenberg, R J
(1990) Herpes simplex vu-us, m Immunochemzstry of Vu-uses, II The Baszs for
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ed ), Elsevier, Amsterdam, pp 45948 1
23 Nicola, A V , Levm, K. P , Willis, S H , Cohen, G H., and Eisenberg, R J
(1994) Studies of HSV glycoprotem D stability. 19th International Herpesvrrus
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24 Rhodes, G (1993) Crystallography Made Crystal Clear, Academic, San Diego
25 Rorzman, B and Sears, A. E (1987) An mquny mto the mechanisms of herpes
simplex virus latency Ann Rev Mlcroblol 41, 543-571.
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M , Cohen, G. H , and Eisenberg, R. J (1988) C3b receptor activity on transfected
cells expressing glycprotem C of herpes simplex vu-us types 1 and 2 J Vzrol. 62,
4027-4036
27 Sisk, W P , Bradley, J D , Leopold, R J , Stoltzfus, A. M , Ponce de Leon, M ,
Half, M., Peng, C., Cohen, G H., and Eisenberg, R. J (1994) High level expres-
sion and purification of secreted forms of herpes simplex virus type 1 glycopro-
tem gD synthesized by baculovuus-infected insect cells J Vzrol 68, 766-775
28. Sodora, D L., Cohen, G H , Muggeridge, M. I , and Eisenberg, R. J (1991)
Absence of asparagme-lurked ohgosaccharides from glycoprotein D of herpes
simplex vuus type 1 results m a structurally altered but biologically active pro-
tem J Vwol 65,4424-443 1
29. Spear, P. G. (1993) Entry of alphaviruses into cells. Semrn Vzrol 4, 167-180
30 Summers, M D and Smrth, G E. (1987) A manual of methods for baculovrrus
Texas Agricultural Experiment Station and
vectors and insect cultureprocedures
Texas A&M University, Bulletin No. 1555, College Station, TX
31 Tal-Singer, R , Peng, C , Ponce de Leon, M., Abrams, W R., Banfield, B W ,
Tufaro, F., Cohen, G H , and Eisenberg, R. J. (1995) The interaction of herpes
simplex vu-us glycoprotem gC wrth Mammalian cell surface molecules J Vzrol
69,447 l-4483
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Enhanced secretion from insect cells of a foreign protein fused to the honeybee
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33. Wall, J. S and Hamfeld, J F. (1986) Mass mapping with the scanning transmrs-
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34 Willis, S. H , Rux, J. J , Burnett, R. M., Ponce de Leon, M., Cohen, G. H , and
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shop, Vancouver, Brrtrsh Columbia, Abst # 13
Crystallization of Macromolecules
for Three-Dimensional Structure Determination
Ben Luisi, Marie Anderson, and Graham Hope


1. Introduction
The last decade has seen a remarkable flourishmg of the biological structure
field. This blossommg has brought an explosion of stereochemical informa-
tion, and has been made possible by the combined improvement in techniques
of X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy,
and the bulk preparation of biological materials. Most crucial, however, has
been the desire of the experimental biologists to follow research problems to
the level of stereochemistry, which is the ultimate reducttontst limit of molecu-
lar btology. This aim has been driven by the anticipation that such knowledge
may permit better understanding and even engineering of biological function.
Structural information has indeed proven mvaluable m functional studies and
has provided a foundation for the interpretation of results from drrected mutagen-
esis. Structural knowledge has often provided clues regarding enzymatrc mecha-
nisms even before functional studies have identified catalytrc centers (2) One
engineering aspectof structural studies, still in its infancy, 1sthe field of rational
drug design. It seemsreasonable that structural information will permit the design
of tailored therapeutic compounds, including molecules, which bmd defined
DNA targets with high specificity and of ribozymes, which, it is hoped, may
selectively inactivate defined RNA species (2,3). Three-dimensional structural
mformation coupled with improvements in the evaluation of molecular
interaction energies may provide better algorithms for rational design programs.
The methods of structure determination by X-ray crystallography and NMR
spectroscopyhave grown increasingly more sophisticated,and it is now possible to
tackle problems that would have been considered impossible a few years ago. In
NMR, signal broadenmg placesa practical upper limit on the sizeof molecules that
From Methods m Molecular Medicme, Vol 10 Herpes Sunplex Vm˜s Protocols
Edlted by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ

157
Luisi, Anderson, and Hope
158

can be studied, and the molecules must be well-behaved m solution and
monodispersed. If these conditions are sattsfied, the solution structure may be
determined fairly qutckly. For crystallography,the practtcalupper limit is enormous.
Even large viruses have beensuccessfullycrystallizedand studied at high resolution
The rate-hmrtmg stepsfor the success the method, however, arethe capricious acts
of
of growing crystalsand suitably doping them with electron-rich atoms.
Here, we describe some practical methods for the preparation and evalua-
tion of macromolecular crystals for 3D structure determination by X-ray
crystallography We also discuss preparation of specimens for NMR and elec-
tron mtcroscopy. The methods for growing crystals for X-ray diffraction are
extremely simple; however, the requirement for specimen purity 1sexacting.
Consequently, we devote a special drscusston (Section 4.) of the attention
required for specimen preparation and some useful procedures that might some-
times help m obtaining crystals.
2. Materials
2.7. Protein Preparation
It is crucial that the protein samples are of the highest possible purity and
readily available in milligram quantmes. Section 4. provides comments on the
evaluation of protein purity. Preferably, the protein samples are at a concentra-
tion of at least 3 mg/mL in distilled water Most proteins will prefer the presence
of some salt or buffer, and others might require glycerol (also as a cryopro-
tectant for frozen storage) or traces of detergent. Each case will be different,
and experimentation will be required
2.2. Nucleic Acid Preparation
If preparing synthetic DNA for cocrystallizatrons, it is most convenient to
start with tritylated material. Purtfication and detrrtylation can be performed
with a NENSORB reverse-phase column (DuPont). The purified material is
lyophtlized and should be checked for quality by analytical reverse phase
HPLC, gel electrophoresis, or mass spectrometry The dried material is then
dialyzed against 100 mM NaCl and then drstilled water to yield the sodium salt
Centricon concentratmg units (Amicon) are very convenrent for the dialysis and
concentration of the samples. Joachimiak and Sigler (4) make several sugges-
tions for destgmng oligonucleotides to be used in cocrystallizations with proteins.
2.3. Buffers, Precipitating Agents,
Crystallization Wells, and Cover Slips
Table 1 summarizes the precipitatmg agents commonly used for crystalliza-
tion experiments. Table 2 summarizes the most commonly used buffers. Crys-
Table 1
Frequently Used Precipitating Agents for Proteins
Salts
Ammonium citrate
Ammonium sulfate
Lithmm sulfate
Sodium chloride
Sodium bromide
Sodmm/potassmm phosphate
Sodiurn/potasslum citrate
Alcohols
2-Methyl-2,4-pentanedlol (MPD)
2,6-Hexanedlol and 1,2-hexanediol
Isopropanol
Ethanol
Propanol
Long-cham polymers
Polyethylene glycol (PEG), typical mol wt 400-20,000 Dalton
PEG methyl ether, typical weight range
Special additives (usually used at mM concentrations)
Reducing agents (dlthlothreltol, mercaptoethanol, dlthlonite)

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