Herpes simplex virus ribonucleotide reductase: expression in Escherichia coli and purification to homogeneity of a tyrosyl free radical-containing, enzymatically active form of the 38-kilodalton subunit.

0022-538X/89/093769-08$02.00/0

Copyright

© 1989, American

Society

for Microbiology

Herpes

Simplex

Virus Ribonucleotide

Reductase: Expression

in

Escherichia

coli and Purification

to

Homogeneity of

a

Tyrosyl

Free

Radical-Containing,

Enzymatically Active

Form

of

the

38-Kilodalton

Subunit

ROLF

INGEMARSON,1*

ASTRID

GRASLUND,1

ALLAN DARLING,2 AND LARS THELANDER1

Department

of Medical Biochemistry

and

Biophysics,

University

of

Umead,

S-901

87 Umea', Sweden,1 andMedical

Research

Council

Virology

Unit,

Institute

of

Virology,

Church

Street,

Glasgow

Gl

SJR,

United

Kingdom2

Received30January1989/Accepted 16May 1989

Infection of

mammalian cells with herpes simplex virus (HSV) induces a virus-encoded ribonucleotide

reductase which

is

different from

the cellularenzyme.This essential viralenzymeconsists oftwononidentical subunits

of 140

and

38 kilodaltons

(kDa) which have notpreviously been purifiedtohomogeneity. The small subunit of

ribonucleotide

reductases

from

other species containsatyrosylfree radical essential for activity. We

have cloned the gene

for

the small subunit

of

HSV-1

ribonucleotide reductase intoa tac expression plasmid

vector.

After

transfection of Escherichia

coli, expression of the 38-kDa proteinwas detected in immunoblots

witha

specific

monoclonal antibody. About 30

,ug

of proteinwasproducedperliter of bacterial culture. The

38-kDa

protein

was

purified

to

homogeneity

in analmost

quantitative

yield by immunoaffinity

chromatogra-phy. It

contained

a tyrosyl

free

radical which gave a

specific

electron paramagnetic resonance spectrum

identical

tothatwehave observed in

HSV-infected mammalian

cells and clearly

different

from that produced

by

the E. coliand mammalian

ribonucleotide

reductases. The

recombinant

38-kDa subunit

had

full activity

when

assayed

in

thepresence

of HSV-infected

cellextracts

deficient

in the native

38-kDa

subunit.

Ribonucleotide reductase (EC 1.17.4.1) reduces all four

ribonucleotides

to

the

corresponding

deoxyribonucleotides

(34).

The enzymes from

Escherichia coli

and mammalian

cells,

aswellasthoseencoded

by

certain

bacteriophages

and

viruses, have been shown

to

be composed of

two

noniden-tical subunits

which

show similarities between the

different

species.

The E. coli and mammalian enzymes have been

purified

to

homogeneity

andarethe

best-characterized

ones

(38, 40, 41).

In

these,

the

large

subunits contain sites for allosteric effectors and the small subunits havea

tyrosyl

free

radical

interacting

with a

pair

of ferric ions.

Both

the iron

centerand the

radical,

which is stable

only

inthepresenceof the

iron

center, areessential forenzyme

activity

(34).

Mammalian cells

infected with different

herpes viruses,

including herpes simplex

virus type 1

(HSV-1), herpes

simplex

virustype

2, Epstein-Barr virus,

and

pseudorabies

virus, contain

a

ribonucleotide

reductase

activity different

from the

activity

inuninfected cells

(10, 11, 22, 26).

None of these enzymes have been

purified

to

homogeneity.

The HSV-1 ribonucleotide

reductase

consists ofa

large

subunit of 140 kilodaltons

(kDa)

andasmall subunit of 38 kDa

(4, 17,

24). Experiments

withthe

pseudorabies

virus- and the HSV-1-induced enzymes

indicate

that both lack the allosteric

regulation

characteristic of the E. coli and mammalian

en-zymes

(3, 26).

The

pseudorabies

virus enzyme also has a

tyrosyl

free radical which

gives

an electron

paramagnetic

resonance

(EPR)

signal

different from those observed for the

mammalian and E. colienzymes.

The small subunit of HSV-1 ribonucleotide reductase is

encoded

by

a 1.2-kilobase

(kb) transcript,

and the

large

subunit is encoded

by

a5-kb

transcript.

The

transcripts

are

colinear, sharing

thesame3'

end,

butthetranslatedpartsdo not

overlap (28).

The

corresponding

DNA has been

se-* Correspondingauthor.

quenced (13, 28, 29).

We

previously produced monoclonal

antibodies

against

each HSV-1

ribonucleotide

reductase subunit andused themtoshow that theenzymeis builtas a

tight complex

of the

a2P2

type. In

this

complex

the two

subunits,

each

consisting

of two identical

polypeptide

chains,

bind

strongly

to one another

(24).

A

nonapeptide

corresponding

to the

carboxyl

end of the 38-kDa subunit inhibitsenzyme

activity by interfering

with this

binding (9,

14, 31).

The HSV ribonucleotide reductase seems to be essential for virus

growth,

atleast in

nondividing

cells

(7, 19,

32, 33).

Attempts

to

separately

express

enzymatically

active HSV-2

ribonucleotide

reductase subunits in cultured human cells have been

reported, although they

haveresulted inverylow

yields (23).

We have cloned the gene

encoding

the small subunit of HSV-1 ribonucleotide reductase into a bacterial

expression

vector. After transfection of E.

coli, expression

of the HSV-1 38-kDa

protein

wasdetected in immunoblots

with a

specific monoclonal antibody.

The

protein

has been

purified

to

homogeneity

and is

enzymatically

active. It

contains a

tyrosyl

free

radical, giving

a

specific

EPR

signal

which is identical to the EPR spectrum observed from

HSV-infected mammalian cells. This spectrum is

clearly

different from those

arising

fromthe E. coliand mammalian

reductases.

MATERIALS AND METHODS

Plasmids. The

plasmid pSG 124, containing

the 23-kb EcoRI A

fragment

ofthe HSV-1 strain KOS DNA cloned

into

pBR 325,

was

kindly supplied by

M. Levine of the

University

of

Michigan,

Ann Arbor

(18).

The

plasmid pDR

540 is an

expression

vector constructed

by

Russel and

Bennet

(35)

and is

commercially

available from

Pharmacia,

Inc., Piscataway,

N.J. The

plasmid

contains the strongtac

promoter, which is

composed

of the -35

region

of the trp

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3770 INGEMARSON ET AL.

promoter and the -10 region, operator, and

ribosome-binding

site of the

lau

UV-5 promoter. The promoter

is

controlled

by thelactose repressor,

and

transcription

can

be

induced by the addition of

isopropyl-4-D-thiogalactoside.

Plasmid

DNA was prepared

from

overnight cultures of

infected

E.

coli

cells

gently

lysed by

treatment

with

lyso-zymeandthen withTriton X-100.The DNA was

purified by

two

consecutive

CsCl gradient

ultracentrifugations.

Bacterial strains and media. The

plasmid pDR

540

was

propagated

in E.

(oli

K-12

JM109

(43).

Transfection

of

E.

coli

was performed asdescribed by Hanahan (21). Bacteria

were grown in LB mediumat

37°C,

andbacteria containing

plasmids

were grown in the presence of 50 pLgof carbacillin (Astra) per ml.

Extraction of bacteriaforenzymepurification andassay. E.

c0li JM109

cells containing the

38-kDa

protein expression

plasmids weregrowntoanoptical density at590nm

(OD,,)

of

3.0. The cellswerethenpelletedat

4°C,

washedonce in25

mM

4-(2-hydroxyethyl)-1-piperazine

sulfonic acid

buffer,

pH 7.6, suspended in the same buffer to an

OD,9(

of

325,

and

frozen

in liquid nitrogen. After the cells were thawed,

KCI

and phenylmethylsulfonyl fluoride were added tofinal

con-centrations

of80 and 1 mM, respectively. Egg white

lyso-zyme(Sigma ChemicalCo., St. Louis, Mo.) wasadded toa

concentration of 300

[tg/ml,

and the mixture was incubated on

ice

for 20

min.

After another cycle of freezing and

thawing,

cell debriswasremoved bycentrifugationat44,000

x gfor60

min

at

4°C.

Enzymes. Restriction endonucleases werepurchased from

IBI

(BamnHI

and HindIII) and Boehringer GmbH,

Mann-heim,

Federal Republic of Germany) (EcoRl and NcoI). T4

ligase, the Klenow fragment of DNA polymerase, alkaline

phosphatase, and mung bean nuclease all came from

Boeh-ringer

GmbH.

DNA sequencing. A309-base-pair

HindIII

restriction

frag-mentcontainingthepromoterandthefirst nucleotides ofthe

herpesvirus

DNA insert was isolated from the plasmid pRI

10 (see Fig. 1B) and subcloned in M13 mpl9 (30). The

sequence was determined by the dideoxy method (37). The

plasmid

pRI 9 (see Fig.

1B)

was sequenced directly at the

plasmid

DNAlevel by themethod of Chen and Seeburg(8).

A 15-mer oligonucleotide corresponding to the sequence

between

the -35 and -10region of the promoter was used as a primer. The primer was synthesized by Symbicom,

Ume'a,

Sweden.

Antibodies. The mouse monoclonal antibody 535 directed

against

the 38-kDa subunit ofHSV-1 ribonucleotide reduc-tase was purified from ascitic fluid by ammonium sulfate

fractionation

(24) followedby chromatography on aprotein

A-Sepharose

column (Pharmacia). A 5-mg portion of

anti-body

waslinkedto1 mlof CNBr-activated Sepharose4B by

the

methods recommended by the manufacturer

(Pharma-cia).

This column could bind at least 0.5 mg of 38-kDa

proteinpermlof sedimented Sepharose. Polyclonal

antibod-ies

directed against a nonapeptide corresponding to the

sequence ofthe nine carboxyl-terminal amino acid residues

of

the HSV-1 38-kDa subunit wereinduced byinjecting 0.5

mgof peptidelinkedto1.6 mgof hemocyanin (Sigma) intoa

rabbit. The couplingwasmade in 0.1 M NaPO4, pH 8.0, in

thepresenceof 6.7mM glutaraldehyde for1 hat

37°C.

After

equilibration

with 0.1 M sodium phosphate, pH 7.6, on a

SephadexG-25 column, the peptide solution was combined

with an equal volume of complete Freund adjuvant. The

boosting was made with the same amount of peptide in

incomplete Freund adjuvant, and then antibodies were

pu-rified

fromthe rabbit serum by ammonium sulfate

fraction-ation (see above)

followed

by

dialysis

against

0.2

M

sodium

citrate

buffer, pH

6.5.

The

antibodies

were

then

linked

to

CNBr-activated Sepharose 4B as

described above

by

using

9 mg ofantibodies perml

of Sepharose. The

binding

capacity

was around 80

[tg

of 38-kDa

protein

per

ml

of

sedimented

Sepharose. The

nonapeptide

was

synthesized by

M.

Carl-qvist at the Department of

Biochemistry, Karolinska

Insti-tute, Stockholm, Sweden.

Polyacrylamide gel electrophoresis and

immunoblotting.

Sodium

dodecyl

sulfate

(SDS)-polyacrylamide

gel

electro-phoresis was carried out as described

previously

(16).

Pel-leted bacteria were

lysed

at

100°C

for

5

min

in gel sample

buffer consisting

of

0.125 M Tris

chloride, pH 6.8, 0.18

M 2-mercaptoethanol,

1.1%

SDS, and

25% glycerol.

To

reduce

the viscosity of the samples, DNase I was added to a

final

concentration of 0.03

mg/ml,

and

the

mixture

was

incubated

for 5

min

at room temperature. In the

immunoblots,

proteins

were transferred from the gel to a nitrocellulose membrane for 2 h at 130 mA by using a

semidry electroblotter

from

Ancos, Denmark (25). After blocking, the membranes were incubated in a solution containing the mousemonoclonal 535 antibody and then in asolution

containing rabbit anti-mouse

antibodies conjugated to alkaline phosphatase (Sigma) as described previously (24).

Partially purified 140-kDa subunit of HSV-1 ribonucleotide reductase. Partially purified 140-kDa subunit of HSV-1 ribo-nucleotide reductase was obtained from BHK-21 cells in-fected with HSV-1 strain 17

ts1222.

This strain has a ts mutation in the 38-kDa subunit of ribonucleotide reductase and cannot make a functional protein when grown at the nonpermissive temperature (12, 32). The cells were infected at a multiplicity of infection of 10 PFU per cell, incubated at

39.5°C,

and harvested at 6 h postinfection. After sonication, nucleic acids were removed by precipitation with

streptomy-cin

sulfate

and proteins were precipitated by the addition of ammonium sulfate to

85%

saturation. Finally, the precipitate was dissolved and dialyzed extensively against 50 mM Tris chloride, pH 8.

Protein determinations. The protein concentration in the cell extracts was determined by the Coomassie brilliant blue method of Bradford (6), using bovine serum albumin as a standard. The concentration of the 38-kDa subunit in bacte-rial extracts was determined after immunoprecipitation with an excess of Sepharose-linked 535 antibody followed by SDS-polyacrylamide gel electrophoresis of the dissolved precipitate. After electrophoresis, the gels were stained with Coomassie brilliant blue and the 38-kDa protein bands were measured with a laser densitometer (LKB Instruments, Inc., Rockville,

Md.),

using known amounts of bovine serum albumin as standards.

Assay of HSV-1 ribonucleotide reductase. Ribonucleotide reductase activity was determined by measuring the reduc-tion of

[3H]CDP

as described previously (16) by using the following incubation mixture. Protein, 15 nmol of

[3H]CDP

(Dupont, NEN Research Products, Boston, Mass.; specific activity, 128,000 cpm/nmol), 1.5 pmol of

MgCl,,

1.5

Vxmol

of dithiothreitol, 3 nmol of

FeCI3,

and 6 pLmol of

4-(2-hydroxy-ethyl)-1-piperazine

sulfonic acid buffer, pH 7.6, were incu-bated in a final volume of 150

jtl

for 30

min

at

37°C.

One unit of ribonucleotide reductase activity is defined as the amount of enzyme or subunit which, in the presence of the other

subunit,

catalyzes the formation of1 nmol of dCDP per

min

at 370C.

EPR measurements. A general background for the use of this technique is given in reference 26. A bacterial extract containing

120

p.g of the 38-kDa HSV-1 ribonucleotide

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A HERPES DNA

Ncol

site BamHlsite

PLASMID VECTOR

tac BamHl

CATO. T .

_ 1.7kB

_0

Digestwfith

I--- Bam Hl

ligation of BamHl ends fiN in wfth Klenow enzyme ! ligationofbluntends

B S/D S/D met

Plasmid pRI 10 .CACAGGAAACAGGATCCATG.

of

\\" " 9 .CACAGGAAACAGGATCGATCCATG.

AmSL

pRI10

5.7 kB

BamHl

FIG.

1. (A)Constructionof the expression vector pRI 10. The entire 38-kDa subunit was encoded within a 1.7-kb Ncol-to-BamnHI HSV-1 DNAfragment (HERPESDNA). Thefirst deoxynucleotide C and the start codon ATG are shown. The translation stop codon TGA is shown 1kbdownstream. This insert was ligated to the expression plasmid vector pDR 540 by using theBacmHlrestrictionendonuclease site located 5basepairsdownstream from the tacpromoter. Both the Ncol site and the

BainiiHI

site happenedto be recreated in theblunt-end ligation

atthe 5' end of the insert. The resulting plasmid was named pRI 10. (B) Deoxyribonucleotide sequences of the initiation sites for protein

synthesis intwo differentplasmids. The Shine-Dalgarno boxes AGGA and the start codons ATG are underlined. pRI 10 is the originally

designed plasmid,andpRI9wasextended 4 nucleotides byfillingin thesticky ends of the originalBanmHIsite situated 5 basepairs upstream from the start codon ATG by using the Klenow fragment of DNA polymerase I and religating. TheBainHIsite at the 3' end of theinsert had

previouslybeen destroyed by mung beannuclease treatment after partial

BanmHl

digestion of the plasmid.

reductase subunit was

incubated

with

200

,ul of sedimented

535

antibody-Sepharose

for

3

h at

4°C. The Sepharose

was

pelleted by centrifugation,

washed once with 50 mM Tris

chloride, pH 7.6,

transferredtoanEPRtube,

resedimented,

and frozen in

liquid nitrogen.

The EPR

experiments

were

performed

inaBruker ER-200spectrometer

equipped

witha

10-in.

(25.4-cm)

magnet and an

Oxford

cryostat

for

low-temperaturemeasurements.

Quantitation

was

made

by

com-parison

of the double

integral

of the EPRspectrum at32 K with

that of

a

Cu2+

solution of known

concentration.

Protein

concentration was determined

by extracting

a known vol-ume of

antibody-Sepharose

with

SDS-sample

buffer fol-lowed

by

SDS-polyacrylamide gel electrophoresis.

RESULTS

Isolation ofaDNAfragmentencoding the 38-kDasubunit of

HSV-1 ribonucleotide reductase and its subcloninginto atac

expression

vector.The38-kDasubunitgeneislocated within the 23-kb EcoRI A

fragment

of HSV-1 DNA

(2.

5,

17).

To isolate the gene, the

plasmid

pSG

124

containing

the 23-kb

fragment

was

digested

with

Bam2HI

and with NcoI which

cuts

just upstream from the

start

codon ATG. The

resulting

1.7-kb

DNA

fragment

was

ligated into

the

BalnHI site

of

the

expression

vector as

indicated in

Fig.

1.

After transfection of

E.

coli JM109, the

nucleotide

se-quence

between the

tac

promoter

and the ATG

start

codon

was

determined in

plasmid DNA from a number of

colonies,

and

a

plasmid

called

pRI

10

had the

expected

sequence

(Fig.

1B).

To

further

testthe

influence of the nucleotide sequence

between

the

Shine-Dalgarno

sequence and

the

ATG

start

codon

on

translation,

this sequence

was

modified

as

indi-cated in

Fig. 1B.

The

resulting

plasmid, pRI

9, contained

an

extra 4

nucleotides

compared

with

pRI

10 and

had

an

AGGA

sequence

located

7

base

pairs

upstream from the

start

codon

(Fig. 1B).

Expression

in E. coli and

immunological

detection of the

HSV-1

ribonucleotide reductase subunit.

Bacteria

containing

plasmid pRI

9 or

pRI

10 were

grown for

4 h

either

in the

absence

or

in

the

presence of

1 mM

isopropyl-4-D-thiogalac-toside

(IPTG)

added

at an

OD9()

of

0.7. The cells were

pelleted

and

lysed,

and the extracts were

analyzed

by

immunoblotting

(Fig.

2). Plasmid

pRI

9

expressed

a

polypep-tide

showing

the same

mobility

as the

38-kDa subunit

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3772 INGEMARSON ET AL.

2

1 2 3

:0

N

[image:4.612.323.559.67.281.2]

0

FIG. 2. Analyses ofextractsfrombacteria containing the 38-kDa subunit expression vector constructs and of extract from HSV-1-infectedmousecells byimmunoblotting with themouse

monoclo-nal 535 antibody (see Materials and Methods). The proteins were

separated ina7.5%polyacrylamide gel. Lanes: 1,extract(0.5mgof

protein) from bacteria containing plasmid with the insert in the

wrong orientation; 2, extract (0.5 mg of protein) from bacteria

containingplasmid pRI 9 withnoIPTG induction; 3,extract(40FLg ofprotein) fromHSV-1-infected mouse3T3 BALB cells (24).

present

in

HSV-1-infected

mammalian cells (Fig. 2, lanes 2

and

3). No band

was

observed in cells

containing

a

plasmid

with the

wrong

orientation of the insert (Fig. 2, lane 1). Cells

containing

the

pRI

9

plasmid showed

a stronger

expression

than the

pRI 10

plasmid.

Furthermore,

the pRI 9

plasmid

showed

strong

expression both in the absence and in the

presence

of IPTG

(data

not

shown). Therefore, the

pRI 9

cells

without IPTG induction

wereused in

all further

exper-iments.

To quantify the

amounts

of the 38-kDa protein in the

E. coli extracts,

cell

lysates were

immunoprecipitated by

using

the

535

Sepharose-linked antibody. After

SDS-poly-acrylamide gel electrophoresis of the dissolved precipitate

followed

by Coomassie

brilliant blue

staining,

protein

con-centration was

determined

as

indicated in

Materials and

Methods.

About 30

,ug of 38-kDa subunit

were

obtained

per

liter of stationary-phase bacterial culture (about 3 OD590 units per

ml).

The 38-kDa

HSV-1

ribonucleotide reductase subunit

pro-duced in E.coli is enzymatically active. As

shown earlier,

the 535

monoclonal antibody does

not

neutralize the activity of

the

HSV-1-induced ribonucleotide reductase

but only binds the 38 kDa subunit

with high affinity

(24).

Therefore,

this

antibody

linked

to

Sepharose

was usedto

bind

and

concen-tratethe

38-kDa

subunit

present

in

a

bacterial

extract made from a

stationary-phase bacterial

culture. After a wash to

remove

unbound protein,

the ribonucleotide reductase

ac-tivity of the

immobilized

protein was measured in the

presence

of

an extract

from

BHK cells

infected

with the HSV-1 ts1222mutant. Extractsfrom such cellsgrownatthe

nonpermissive

temperature lackedafunctional 38-kDa viral

0 1 2 3 4 5 6

[image:4.612.144.222.69.323.2]

Bacterial extract

(ml)

FIG. 3. Proportionality between increasingamounts ofE.

coli-produced 38-kDa subunit and HSV-1 ribonucleotide reductase

ac-tivity inareconstitution experiment. Increasingamountsofextract

from plasmid pRI 9-containing bacteria were mixed with 20 i,l of

sedimented 535 antibody-Sepharose inaseries of tubes and

incu-batedfor 3hat4°Cunderconstantmixing.TheSepharosewasthen

pelleted by centrifugationandwashedoncein50 mM Trischloride, pH 7.6.Immediately beforetheassay wasperformed,300Fgof the

ts1222-infected BHK cell extract wasadded to each tube. A1-ml quantity of bacterial extract corresponds to 100 ml of bacterial

culture(2

OD590

unitsperml).

ribonucleotide

reductase subunit

(12).

The results from

experiments

inwhich

increasing

amountsofa

plasmid pRI

9

bacterial extract were incubated with an excess of 535

antibody-Sepharose

followed

by

the addition of aconstant amount of ts1222-infected-cell extract are shown in

Fig.

3. The enzyme

activity increased

rapidly

with

increasing

amounts

of

bacterial extract, but then the increase became

slower,

which indicated that the

large

subunit is

limiting,

since the 535

antibody-Sepharose

was still far from

satura-tion. No

activity

wasobserved when

comparable

amountsof each subunitwere

assayed separately

(Table 1).

The

specific

activity

of the immobilized 38-kDa subunitwas 1.4

U/mg.

Purification of HSV-1 38-kDa

ribonucleotide

reductase sub-unit

produced

in E. coli.

Unfortunately,

the 535

antibody

bindstoo

strongly

tothe38-kDa subunitto allowelution of thebound

protein

inanactiveform

(24).

For the

purification

we instead used an

affinity column

containing

Sepharose-TABLE 1. Ribonucleotide reductaseactivityobtainedbymixing the38-kDa subunit produced in E. coli with the 140-kDa subunit

presentints1222-infected BHK cells

Antibody-

Free

38-kDa s1222-infected Enzyme Sp.act.

Sepharosebound ,, BHKcell

38-kDasubunit(k,g) subunit (p.g) extract(p.g) activity (mU) (U/mg)

3.5 350 4.8 1.4

3.5 0

350 0

0.1 350 1.5 15.0

0.3 350 2.8 9.3

0.5 350 3.7 7.4

"Containing0.44 mgof bovineserumalbumin per ml as a carrier.

J. VIROL.

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1

2

Mr

116

_io

66

46

36

29

FIG. 4. SDS-polyacrylamide gel electrophoresis of the 38-kDa HSV-1 ribonucleotide reductase subunit produced inE. coli after elution from polyclonal antibody-Sepharose. The gel contained 7.5% polyacrylamideandwasstained withCoomassie brilliantblue. Lanes: 1, 1.3 jig of the eluate analyzed after precipitation with trichloroacetic acid(16): 2. molecularweight markers

(3-galactosi-dase [116 kDa], bovine serum albumin [68 kDa], ovalbumin [46 kDa], glyceraldehyde-3-phosphate dehydrogenase 136 kDa]. and carbamic anhydrase [29 kDa] [Sigma]).

linked rabbit polyclonal antibodies directedagainst the

car-boxyl-terminal nonapeptide of the 38-kDa subunit. This nonapeptide (YAGAVVNDL) is quite different from the

carboxyl-terminal sequence of the small subunit of the E.

coli

ribonucleotide reductase and wasalso used toelute the

38-kDa subunit from the column.

Acrudeextractfrom 7.3 liters ofstationary-phasebacteria

containing the pRI9 plasmid was passed through a 2.7-ml

antibody column at4°C.Thecolumnwaswashed with30ml of 50 mM Tris chloride,

pH 7.6,

5.4 ml of the same buffer

containing 0.5 MKCI, and 7 ml of 50 mM Tris

chloride,

pH 7.6, all at 4°C. The column was then moved from the cold

room to room temperature and was immediately washed with 6ml of

carefully degassed

50 mM Tris

chloride, pH

7.6.

at

25°C. Finally,

bound

protein

was eluted in 3.5 ml of the

same buffer containing 1 mM nonapeptide at 25°C. An

aliquot of the eluate was analyzed by SDS-polyacrylamide

gel electrophoresis (Fig. 4). Only one protein band was

observed, and this

migrated

at 38 kDa (lane

1),

but laser

densitometry scanning

revealedanadditionalveryfaint band

at around 80 kDa which possibly was the dimer. After the

immunoaffinity chromatography

step, 113 p.g of 38-kDa

protein was obtained, resulting in a recovery of 70%. This

was estimated from immunoprecipitation of the bacterial

extract and SDS-polyacrylamide gel electrophoresis of the eluate. Direct measurement ofprotein concentration in the eluatewas prevented by the presence of thenonapeptide.

The

nonapeptide

is known to

inhibit

enzyme activityand

therefore

had to be removed. Direct fractionation with ammonium sulfate could not

be used

because the

nonapep-tide precipitated together with the protein. Instead,

bovine

serum albumin was added as a carrier protein to the eluate (0.7

mg/ml),

and then the solution was passed through a

Sephadex G-50

column (sample volume to column

volume

ratio,

1:10) equilibrated with 50 mM Tris

chloride,

pH 7.6. This gave a quantitative recovery of the 38-kDa protein

and

a clear separation from the

peptide.

The concentration of 38-kDa protein in the Sephadex G-50 eluate was determined ina

portion by SDS-polyacrylamide

gel electrophoresis,

and

the rest of the solution was

kept

frozen at -70°C.

The

specific activity

of the purified 38-kDa subunit was

measured

in the presence

of large subunit

from

ts1222-infected

cells, giving

a

value of 15 U/mg (Table

1). No activity was

observed when either

the 38-kDa subunit or the t.s1222 extracts were assayed separately. For comparison, a

partially purified

HSV-1-infected Vero cell extract assayed in

parallel

had aspecific activity of 0.04 U/mg. Furthermore, a homogeneous preparation of the small subunit ofmouse

ribonucleotide reductase

had a

specific activity of

55 U/mg

when assayed

inthe presence of a large excess of pure large

subunit (41).

In our assay,

the

activity did

not

increase in

a linearway when the amount ofpurified 38-kDa subunit was

increased

in the presence

of

a constant amount of the large subunit

(Table 1).

This indicated that the amount oflarge subunit in

the

ts1222-infected

cell extract was

limiting

in the assay.

EPRspectroscopyof the 38-kDa subunit of HSV ribonucle-otide reductase

produced

in E.coli. An EPR spectrum at

32

K of 535

antibody-Sepharose-linked

38-kDa subunit is shown

in

Fig.

Sa.

For

comparison,

the

corresponding

spectra

of the

tyrosyl

free radicals of the small subunits of ribonucleotide

reductase

in

HSV-1-infected Vero cells (Fig. Sb), in

mouse fibroblast cells

(Fig. Sc),

and in E. coli cells

(Fig. Sd)

are

shown.

For

each

EPR spectrum, the

overall

spectral shape

reflects the

hyperfine interactions of the radical.

In our

interpretation

(20), differences in hyperfine

structure among these

radical

spectra are to a

large

extent

dependent

onthe

angle between the

3-methylene hydrogens of the

tyrosine

and the

plane

of its

aromatic

ring.

In

this

respect,

the

EPR

signal from

the

recombinant

38-kDa

protein

is identical

to

the

EPR

signal

in

HSV-infected Vero cells but

is

clearly

different from the

signals originating from

the noninfected

mammalian and

E. coli

cells.

When the

microwave

satura-tion behaviors

of the

signals

are

compared,

the HSV-1

ribonucleotide reductase produced in

E.coli

is

very

different

from the

ordinary

E.

coli enzyme

and is in fact

more

like the

mammalian

one

(36).

By

quantitation

of

the EPR

signal,

the

concentration of

the

tyrosyl free radical in

the

Sepharose-bound

38-kDa

protein

was

estimated

to be

3

p.M.

This

value

did not

increase after

incubation with iron-dithiothreitol,

a treatment

known

to

regenerate

the radical

in the

mammalian

small

subunit of

ribonucleotide reductase

(41).

By

using

a

molecular

weight

of

76,000,

the

dimer

concentration of the 38-kDa

protein

in the EPR tube was estimated to be 7.9

F.M,

which

corre-sponds

to0.4 radicals per dimer.

DISCUSSION

Because

of

the

tight

intersubunit

binding

in the HSV

ribonucleotide

reductase

(24)

and the low abundance of enzyme, it hasnotbeen

possible

to

purify

the

individual

140-and

38-kDa subunits

to

homogeneity

from

HSV-infected

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[image:5.612.145.212.62.338.2]

3774 INGEMARSON ET AL.

FIG. 5. EPR spectra at 32 K and nonsaturating microwave

powerconditions of the following substances. (a)A0.12mgsample of HSV-1 38-kDa ribonucleotide reductase subunitproduced in E.

coli and immobilized in 535 antibody-Sepharose. The microwave

power was 3.9 mWand the modulation amplitude was 2.0G. (b)

HSV-1-infected Vero cells. Confluent monolayers of Vero cells

wereinfected with HSV-1 at amultiplicity of infection of 10, and

after 9.5 hat37°C the cellswereharvested,packed inanEPRtube,

frozen, and stored in liquid nitrogenas described in reference 26.

The EPRspectrometer conditions wereessentially the sameas in panela. (c)Hydroxyurea-resistantmousefibroblast 3T6 cells which

overproduced the small subunit of mammalian ribonucleotide

reduc-tase (1). (d) E. coli KK546 cells which overproduced the E. coli ribonucleotide reductase (39).

cells.

The

apparent

lack of allosteric regulation (3), the

presence

of

a

large subunit of 140 kDa compared with

one

of

around

90 kDa

in

other species (34) and

thevery

interesting,

highly specific protein-protein interaction observed between

the

140-kDa

subunit

and

a

nonapeptide corresponding

to

the

C-terminal sequenceof the small subunit

(31)

made it

desir-able to obtain

sufficient

amounts

of

pure subunits to

allow

further studies.

We have therefore used a tac

expression

vector to

pro-duce the 38-kDa

subunit

in E.

coli cells. Our best DNA

construct

expressed around

30 p.g

of protein

per

liter of

culture,

and this

expression

was

independent of IPTG

induc-tion.

E.

coli lacks the

posttranslational machinery of

mamma-lian

cells. The 38-kDa protein

produced in E.

coli

had the

same

mobility in SDS-polyacrylamide

gel electrophoresisas

the

corresponding protein from HSV-infected

Vero cells. This

fact together with

the

enzymatic activity

of the

recom-binant

protein strongly

argues

against

anymajor

posttrans-lational

modification of

the protein in mammalian cells. There are

data in the literature

suggesting that the HSV

ribonucleotide reductase

subunits are phosphorylated in

infected cells (5, 27,

42),

but

the

significance

of these findings

ought

tobe

further

studied.

Wecannotexclude thepresence

ofa

protein kinase in the partially purified

140-kDa subunit

preparation from HSV-1

ts1222-infected cells. However,

since no ATP

was

present

during the assay to serve as a

phosphate donor, such an enzyme would not be able to

phosphorylate the 38-kDa

protein.

The

38-kDa

protein produced in

E.

coli

was

HSV

specific

and

was

distinguished

from

potentially contaminating E.

coli

ribonucleotide reductase

B2

subunit in the

following

ways.

Enzyme

activity

was

obtained in reconstitution assays with

the

38-kDa subunit

immobilized

to

the

535 monoclonal

antibody-Sepharose;

this

antibody

has

no

affinity

for the E.

coli

B2

protein.

Furthermore,

activity

was

completely

de-pendent upon

the

addition of the HSV-specific 140-kDa

subunit.

Finally,

the EPR

spectrum

of the recombinant

38-kDa

protein

was

identical to the spectrum of the

ribonu-cleotide reductase small subunit present in HSV-infected

Vero cells

(shown for the first time in this paper). This

spectrum

is

completely

different from the

corresponding

spectra

of the small subunits of ribonucleotide reductase in

E.

coli and mammalian cells (36, 39, 41) in both

shape and

saturation

behavior.

Despite the activity of the

antibody-immobilized

38-kDa

subunit,

we

assumed that

a

molecule free

in

solution without

any

steric hindrance would be

more

active.

Therefore,

the

38-kDa

protein

was

purified

to

homogeneity by

immunoaf-finity chromatography. After removal of the

eluting

non-apeptide, the 38-kDa

protein

was

almost

as

enzymatically

active

as

the pure M2 subunit

of mammalian

ribonucleotide

reductase. The

free 38-kDa

protein

was

about 10 times

as

active

as

the

antibody-immobilized protein (Table

1). The

specific activity

was

dependent

on

the

amount

of

partially

purified

140-kDa subunit present in the assay,

in that

higher

specific activity

was

observed with

decreasing

amounts

of

the

38-kDa

protein.

A

very

interesting

observation is that the 38-kDa HSV

subunit

was

able

to

generate

its

tyrosyl free radical in

E.

coli.

Our data indicate the presence of about

0.4

tyrosyl

free

radicals per 38-kDa dimer.

Considering

the accuracy

of the

protein

determinations and the

possible loss of free radical

during

the

extraction of the

bacteria, binding

to

antibody-Sepharose, washing,

and

freezing, this figure is close

to

the

maximal value of

1

free

radical per dimer determined for the

pure

E.

coli B2

protein

(38). The extraction was made in the

absence of iron-dithiothreitol to prevent radical

generation

outside the bacteria. There

are

data

indicating

a

specific

tyrosyl

free radical

regeneration

system

in

E.

coli (34).

However,

since the amino acid sequence

similarity between

the

E.

ccli B2

protein

and

the HSV-1 38-kDa

protein is

not

very

pronounced (15) (optimal score,

61

[determined by

using

the

amino acid

align

program

of

DNA

STAR

Inc.]

compared

with

scores

of

121

for

B2 versus

the

M2

subunit of

the

mammalian ribonucleotide reductase and 316 for the

38-kDa

protein

versus

M2),

we

find it

more

likely

that the

radical formation in

the

38-kDa

protein is

an

intrinsic

prop-erty

of the

protein

itself

once

it is

supplied

with

ferrous iron

and oxygen. The

environment inside

E.

coli fulfills this

requirement.

This convenient and reproducible way of preparing

the

38-kDa subunit of HSV-1

ribonucleotide reductase resulted

in

20 to 30

p.g

of pure

protein

per liter

of bacterial culture and

should enable

more

detailed

studies of this very

interesting

and

medically important

form of

ribonucleotide

reductase.

ACKNOWLEDGMENTS

This work was supported by grants from the Swedish Natural Science Research Council, Magn. Bergvalls Stiftelse, the Kempe J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.612.79.279.68.328.2]

Foundation, the Medical Faculty at the University ofUme'a.and the Medical Research Council of Great Britain.

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