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5 1X phosphate-buffered saline (PBS). 140 mM NaCl, 2 7 mA4 KCl, 10 n-u!4
Na,HPO,, 1 8 mA4 KH,PO, (pH 7.4)
6. 6X SDS loading buffer 0 35MTrts-HCl (pH 6 S), 10.28% (w/v) SDS, 36% (v/v)
glycerol, 5% P-mercaptoethanol, 0.012% (w/v) bromophenol blue

2.4. Miscellaneous Equipment
Orbital shaker with variable temperature control, microfuge, benchtop centri-
fuge (e.g., Fison™s coolspm), high-speed centrifuge with SLA-3000 rotor and
bottles (or equivalent), SO-mL disposable plastic tubes, apparatus for sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reagents for
fixing gels, and staining with Coomassieblue.
3. Methods
3.1. Generation of an Expression Vector
Containing the DNA Sequences of Interest
A fragment of DNA encoding the chosen polypeptide can be obtained either
by purification of a restriction enzyme fragment from an agarose gel or by
PCR. Generation of fragments by PCR for subclomng into expression vectors
has the advantage that the primers can be designed to incorporate specific
restriction enzyme recogmtion sites, so that the gene of interest can be cloned
directionally mto available restriction sites in the vector. In the case of fusion
proteins especially, the recombinant plasmid should be sequenced across the
junction between the fusion partner and the gene of interest to ensure that the
protein has been cloned m the correct reading frame. This is particularly import-
McKie
188
ant when the expressed protem is not the expected size as predicted from its
amino acid sequence.
The recombmant plasmid is used to transform an appropriate E colz strain.
A glycerol stock can be prepared by picking a transformed colony into 5 mL
2X YT containing 250 ug/mL ampicillm and growing overnight with shaking
at 37OC. An equal volume of glycerol is added before ahquotmg mto 1 mL
amounts and storing at -70°C.
3.2. Small-Scale Production of Fusion Proteins
Small cultures of bacteria contaimng the fusion protein of interest can be used
to determine the optimal factors for expression of soluble fusion proteins in terms
of length of induction period and temperature of induction. As a control, bacteria
transformed with the parental plasmid vector containing no insert should be used.
Day 1: Scrape a small amount of bacteria from your glycerol stock mto 1
mL LB. Plate out 100 uL onto an LB agar plate. Incubate in an inverted posi-
tion at 37OC overnight
Day 2:
1, Pick a transformed bacterial colony from the agar plate into 5 mL LB (170 mMNaC1,
10 g/L Difco bactottyptone, 5 g/L yeast extract) containing 100 ug/mL. ampicillin
2 Grow at 37°C for 3-6 h with shaking until an A,,, of (0.60 8) is reached
3 Induce fusron protein expression by the addition of 50 uL of 100 mM IPTG
4. Remove 500˜)rL samples at hourly intervals, and pellet the bacteria in a microfuge
by spinning at 6500g for 5 min.
5 Resuspend in 250 uL of 1X SDS gel loading buffer and boil.
6. Analyze 15-uL (mmlgel), or 50-pL (maxigel) ahquots by SDS-PAGE.
Gels can be fixed and stained with Coomassie blue. It should then be pos-
sible to visually determine the length of mduction time required for optimal
protein expression. Stmilar experiments can be carried out to examine the effect
of variations m the concentration of IPTG used for induction on protein
expression, smce lower or higher concentrations than those given above may
be optimal for expression of some proteins. Having assessed the optimal
induction times and IPTG concentrations, production can now be scaled up to
determine the solubility of the expressed protein.
3.3. Determining the Solubility of the Expressed Protein
To determine the solubility of the expressed protein.
1. Inoculate several 5-mL cultures with a single transformed bacterial colony as
described in Section 3.2 , and induce with IPTG for the optimal determined time.
2 Pool the 5-mC cultures, and centnfige at 2000g for 10 mm in a Fison™s coolspm at 4°C
3. Resuspend the pellet in 5 mL PBS containing 500 ug/mL lysozyme, and incubate at
room temperature for 10 mm. Fully lyse the bacteria on ice using a probe sotucator
189
Herpes Simplex in Bacteria
Note: The tip of the soniprobe should never be completely immersed into the
tube. Instead, it should be placed Just below the surface of the solution. Care
should be taken not to allow frothing of the solution as thts is indicative of
oversonication, which may lead to aggregation or denaturation of the proteins.
4. Spin at 2000g for 10 mm at 4°C in a Fison™s coolspin to pellet the bacterial debris
Pour off the supernatant and retain. Resuspend the bacterial pellet in 5 mL PBS
Fifty-microliter ahquots of bacterial pellet and supematant fractions can be added
to 10 pL 6X SDS loading buffer and analyzed by SDS-PAGE. If the recombinant
protein is soluble, it should be present primarily in the supernatant fraction; if tt
IS insoluble, it will be present in the fraction that contains the bacterial debris.
3.4. Large-Scale Production of Crude Bacterial Lysates.
After carrying out small pilot experrments to establtsh optimal conditions for
expressron, a large-scale purification can be performed. Small samples should be
retained at each step in the procedure for analysis of the purification method.
1 Inoculate a transformed bacterial colony into 100 mL of LB containing 100 pg/
mL ampicillm, and incubate with shaking at 37°C overnight.
2. Dtlute this culture m 900 mL LB + amptctllm, and split between two 2 or 3 L
flasks. Grow at 37°C for 3 h.
3. Add IPTG to induce fusion protein expression, and continue mcubatton for the
time required to obtain maximal expression.
4 Pellet bacteria by spinning at 5000g for 10 mm m an SLA-3000 rotor, and resus-
pend m -50 mL of PBS containing 500 ug/mL lysozyme Split sample evenly be-
tween two 50-mL Oakrrdge tubes. Incubate at room temperature for 1O-l 5 min.
5 Place the bacteria in a container on ice, and sonicate using five or six 15-s bursts to
lyse the cells completely. Following sonication, a detergent solution, e.g., 500 pL
Tween-20, could be added to the lysate to aid solubilization of the fusion protein.
6. Centrifuge at 10,OOOg for 15 min m an SS34 rotor to remove bacterial debris.
7 Collect the supematant, which 1sthe crude bacterial lysate, for further purtficatton.
The next stage in purification will be dependent on the type of expression
vector used. For purification of native full-length recombmant proteins,
ammonium sulfate purification 1s commonly used.
3.5. Ammonium Sulfate Fractionation of Expressed Proteins
Ammonium sulfate fractionation can be utilized to purify expressed pro-
teins partially from bacterial extracts. This mvolves adding increasing amounts
of a saturated solution (4M) of ammonium sulfate to crude bacterial extracts
and determining by SDS-PAGE the optimal concentration for precipitation of
the expressed protein.
1. Add increasing amounts of ammonium sulfate to 100-PL aliquots of crude bacte-
rial extracts in Eppendorf tubes, and Incubate for 30 mm on Ice. Table 1 shows
the volumes of ammonium sulfate that are required to give final concentrations
McKie
190

Table 1
Precipitation of Crude Extract
Using a Saturated Solution of Ammonium Sulfate
Volume 4M solutron
Fmal % ammonium
Volume crude extract, pL sulfate added, pL
100 5 53
100 10 11 1
100 15 17.7
20 25.0
100
25 33 4
100
100 30 42.8
35 53.9
100
40 66.7
100
100 45 81.9
100 50 100 0


m the range of 5-50% when added to 100 pL crude extract
2. Spin samples at 13,500g for 30 mm in a microfuge at 4T.
3 Redissolve pellets m 300 pL PBS, and bring supernatants up to an equal volume
4 Analyze pellet and supernatant fractions by SDS-PAGE
pellet and supernatant fractions over a range of ammomum
By comparing
sulfate concentrations, tt should be possible to determine the ammonium sul-
fate concentration at which the maJorlty of the expressed protem 1sprecipitated
from solution.
For large-scale ammomum sulfate fractionation, ammonium sulfate powder
is slowly added to the extract with constant stirring, at room temperature, fol-
lowed by incubation on ice for at least 30 min. The protein precipitate IS col-
lected by centrtfugation at 12K for 30 mm m an SS34 rotor at 4°C. The pellet IS
then resuspended in PBS. Further purification will probably involve techniques,
such as FPLC ion-exchange chromatography.
3.6. Purification of GST Fusion Proteins
GST fusion proteins are easily purified from bacterial lysatesusing glutathione
Sepharose4B that has been equilibrated using PBS. Glutathlone Sepharose4B IS
supplied from Pharmaciaas a 75% slurry. It should be washedtwice using 10
mL of PBS, and resuspended m 1 mL PBS for each 1.33 mL of glutathione
Sepharose used. This results m a 50% slurry. One mrlhliter of a 50% slurry is
4B
generally sufficient to purify recombmant protein from 1 L of bacteria.
1 Add supernatant from 1 L of bacterial extract to 1 mL of 50% slurry of glu-
tathrone Sepharose 4B beads, and mix gently m a rotating wheel at room tem-
Herpes Simplex in Bacteria 191

perature for IO-15 min. Pellet the beads at 2000g for 2 mm m a Ftson™s coolspin,
and gently wash three or four times wtth 50 mL of PBS After the final spin,
resuspend the beads m 1 mL PBS and transfer to an Eppendorf tube
2 Microfuge at 13,SOOg for 1 mm to pellet the beads, and carefully remove the PBS
using a yellow tip
3 Resuspend pellet m 1 mL of 50 mMTris-HCl (pH 8 0)/5 mA4reduced glutathione
Mix on a rotating wheel for 5 mm, and pellet beads as before Remove the super-
natant which contams the eluted fusion protein, and store. Repeat elution step a
further four or five times. Remove small ahquots at each elution step, and ana-
lyze using SDS-PAGE to ensure that the protein is totally eluted from the beads
It 1salso advisable to run a small portton of the beads on the gel to check that no
protein still remains bound While awamng this result, the glutathtone Sepharose
4B can be stored short term at 4°C in 1 mL of 50 mMTrts-HCl (pH 8 0)/5 mM
reduced glutathione.

3.7. Removal of the GST Affinity Tail
tt may be desirable to remove the GST portion of the
In some situations,
expressed protein. GST is a relatively immunogenic protein, so if the fusion
protein that you have produced is to be used for antiserum production, and
your protein of interest is known to be of low immunogenicity, it is better to
remove the GST portion of the expressed protem rf this IS feasible
All pGEX vectors contam thrombin or factor Xa protease recognition sites,
and cleavage occurs most efficiently when the fusion protein 1sbound to the
glutathione Sepharose 4B:
1. Roughly determine the amount of protein bound to the beads using SDS-PAGE.
2. Wash the beads twice with 20 mL of 1% Triton X-100 in PBS, once with 50 m1!4
Trts-HCl, pH 7.5/150 nuV NaCl and once with either thrombm cleavage buffer
(50 mM Tris-HCl, pH 7 5, 150 mM NaCl, 1 mM CaCl*) or factor Xa cleavage
buffer (50 mA4Trts-HCl, pH 7.5, 150 mA4NaCL2.5 mMCaCl&
3. Resuspend beads m -1 mL of the appropriate cleavage buffer containing the ap-
propriate concentratton of either thrombin or factor Xa (refer to manufacturer™s
instructions),and incubate at room temperature for 1 h on a rotating wheel.
4. Pellet beads and collect supematant by spmmng at 13,500g for 1 mm m a mtcromge.
Repeat wash steps three or four times and analyze each fraction using SDS-PAGE

3.8. Commonly Encountered Problems
Fusion protein does not elute from glutathione Sepharose 4B, or elution is
inefficient: It may be necessary to increase the concentration of reduced glu-
tathione used. Up to 15 mM glutathione can be used without any effect on the
pH of the buffer. Alternatively, overnight elution may be required.
Expression levels are low. This problem is often encountered with herpes
simplex virus proteins, and 1s probably owing to the difference in codon usage
McKie
192
between the host bacteria and herpes simplex, which has a high G-C content.
In this case, it is useful to vary the length of induction and concentration of
IPTG used. Delaying the time of induction from 3 h to 5-6 h will give a denser
culture of bacteria to begm with and possibly a higher yield of expressed pro-
tein. Alternatively, if high concentrations of purified protein are required, it
may be necessary to shorten the length of the polypeptide that is bemg expres-
sed or even use a eukaryotic expression system.
Expressed protein is msoluble: There are several ways m which the solubil-
ity of proteins can be improved, the most simple of which is to reduce the
induction temperature. We have found on several occasrons that proteins that
are insoluble when induced at 37°C are more soluble at lower temperatures,
e.g., 30 or 28°C. The addition of detergents, e.g., 1% Triton X- 100, 1% Tween-
20, 10 rnM DTT, and 0.01% CHAPS, may all also aid solubrbzatron. Alterna-
tively, tt may be necessary to express a smaller portion of the polypepttde to
exclude regions that are highly charged or hydrophobic.
Bacterial proteins copurify with the expressed protein: This may be owing
to oversonication of the fusion protein, leadmg to aggregation, and can be pre-
vented by reducmg the somcation time and frequency. If the copurifymg pro-
teins are of a lower molecular weight than the expressed protein, they may be
breakdown products. These can be mmimrzed by decreasing the length of the
induction period and adding protease inhibitors to the medium used for har-
vestmg the crude bacterial extracts.
References
1. Studier, F. W. and Moffat, B. A. (1986) Use of T7 RNA polymerase to direct
selective high-level expression of cloned genes. J. Mol. Bzol 189, 113-130
2. Studier, F. W., Rosenberg, A H., Dunn, J. J , and Dubendorff, J W (1990) Use of T7
RNA polymerase to direct expressron of cloned genes Methods Enzymol 185,60-89
3 Furlong, J., Conner, J., McLaughlan, J , Lankmen, H , Gait, C., Marsden, H. S.,
and Clements, J. B. (199 1) The large subunit of herpes simplex vnus type 1 rrbo-
nucleotrde reductase, expressron m Escherichla colz and purrticatron. V˜oZogy
192,848-85 1.
4. Lankmen, H , McLauchlan, J., Weir, M., Furlong, J , Conner, J , McGamty, A ,
Minstry, A, Clements, J B., and Marsden, J B (1991) Purrficatron and charac-
terization of the herpes simplex vnus type 1 rrbonucleotrde reductase small sub-
unit following expression m E colr J Gen Vwol. 72, 1383-1392
5 Smith, D B., Davern, K M , Board, P G , Tiu, W U , Garcia, E G , and Mitchell,
G. F. (1986) Mr 26,000 antigen of SchlstosomaJaponlcum recognized by rrsrtant
WEHI 129/J mice IS a parasite glutathione S-transferase Proc Nut1 Acad Scz
USA 83,8703-8707
13
In Vitro Systems
to Analyze HSV Transcript Processing
Anne Phelan and J. Barklie Clements


1. Introduction
During lytic virus replicatton, herpes simplex virus (HSV) exhibits a closely
regulated pattern of viral gene expression and of DNA rephcatton, resultmg in
vmon production (2). Broadly, HSV genes can be divided into immediate early,
early, and late categories basedon the kinetics of then expression. The five imme-
diate early genes are expressed in the absence of prior viral protein synthesis
although their expression is stimulated by a viral tegument protein. Two immedi-
ate early proteins are essential for virus replication in vitro and act at the transcrtp-
tional (IEl75) and posttranscriptional (IE63) levels to regulate early and late gene
expression. Throughout mfection, mRNA is synthesizedusmg cellular RNA poly-
merase II, which is modified by the action of an immediate early protein (2).
Mature mRNAs are formed m nuclei by extensive posttranscriptional pro-
cessing of their primary transcripts. Processing events include splicing, for
intron-containing RNAs, and formation of the 5™-cap structure and the mature
3™-end. Only five HSV genes contain introns (3), three of whtch are expressed
from immediate early times, whereas all but one (a latency-associated tran-
script) are polyadenylated. Both polyadenylation and splicing. require a com-
plex coordination of trans-acting protein-protein and protein-RNA
interactions and, for these posttranscrtptional processes,the vnus is reliant on
host cell functions of transcription. During HSV- 1 infection of permissive cells,
viral gene expression and polypepttde synthesis occur against a background of
declining host macromolecular synthesis and inhibition of host DNA synthe-

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