Vol.

167,

No.

1, 1990

28,

February

BIOCHEMICAL

BIOPHYSICAL

AND

RESEARCH

COMMUNICATIONS

Pages

1990

33-39

SPECIFIC IMMUNO-DETECTION OF BENZOATE-PARA-HYDROXYLASE WITH ANTIBODIES RAISED AGAINST SYNTHETIC PEPTIDES KOEN

GERRITSE,

ROBERT

.JACQUELINE

VAN

LANGE, ERIC

MARC

GORCOM,

MARIANNE

SCHELLEKENS,

CLAASSEN

AND

WIM

FASBENDER,

NETTY

ZEGERS,

BOERSMA

Medicar’ Biological Laboratory TNO, P.O. BOX4.5, 2280AA Rijswijk, The Netherlands Received

January

9, 1990

As a model system for the industrial use of fungal cells in the enzymatic conversion of chemicals, the parahydroxylation of benzoate was studied. To increase the amount of benzoatepara-hydroxyhse (BPH, EC 1.14.13.12.) in the cell the gene coding for the enzyme (bphA) was cloned and expressed in Aspergillusniger. Detection of the enzymatic activity of the protein was not reproducible. It was decided to raise an antiserum for immuno-detection purposes. Sufficient benzoate-para-hydrovlase for immunization could not be obtained ; therefore the synthetic-peptide strategy was used. We demonstrate that synthesis of antigenic determinants, can be useful in the production of highly specific reagents for the detection of proteins. The availability of monospecific polyclonal sera opens new possibilities in functional studies and purification of benzoate-parahydroxylase. 01990 Academic Pres*, Inc. Already coat protein peptides

in 1971 a synthetic

peptide

was used for raising antibodies

has become an important

that short synthetic react with

peptides,

the peptides

predicted

and also with

the native

when

a group

immunodominant bodies

to

class specific structures

[3,4]. :Synthetic peptides

Furthermore protein

belongs

applications

is known

might

are found

[2] and when

against the native protein

from the DNA

peptides

protein

to a part of the tobacco

tool in the production

some cases the use of synthetic the

corresponding

from

homologous

could prevent

which

to intact proteins

important

can not be purified

native where

#corresponding

antisera, provided

to those determinants

that the peptides

conformation

the

[2]. In especially

presence

in sufficient

sequence

quantities

Abbreviations: A. niger, Aspergillusniger, BPH, benzoate-para-hydroxylase; haemocyanin; MBS, m-maleimido-benzoyl-N-hydroxysuccinimide-ester; BSA, EDC, I-ethyl-3-(3-dimethylaminopropyl)-carbodiimide.

of the

and that

of anti-native

protein

in the protein.

KLH, keyhole limpet bovine sera albumin;

0006-291X'90 33

[5].

to be used

determinants

as the sequences

of anti-

of vaccines

the DNA

are suitable for production

have a similar

protein,

in the production only

which

of subclass specific

for immunization, as was demonstrated in the current study. It is assumed that proteins contain a limited number of antigenic only peptides

antibodies

they are derived

the development

in those cases in which

the protein

We have shown

base sequence, can induce

proteins

also become

Since then the use of

of specific antibodies.

is even preferable of

[l].

mosaic virus

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

167,

No.

BIOCHEMICAL

1, 1990

Since neither

the antigenic

were

the sequences

known

improve

carrier

to elicit

sequences,

COMMUNICATIONS

structure

on theoretical

anti-peptide

of the BPH

to the antigenic antibodies,

of the BPH

considerations

the peptides

carrier

sequences

were

for peptide

molecules

against the peptides,

that this approach

can be used for immuno-detection

Materials

chosen

short

the selection

the peptides

We demonstrate

which

of the selected

we describe

to couple

conjugates

RESEARCH

protein

alone. linked

To

to an

protein.

In this report methods

BIOPHYSICAL

nor the three-dimensional

for synthesis where

the immunogenic@

immunogenic

protein.

determinants

AND

synthesis, the

and the capacities

which

crossreact

is useful to raise mono-specific

with

polyclonal

of the

the native antibodies

of the enzyme.

and Methods

Peptidesynthesisand conjugation. Selection of the peptides was based on prediction-plot patterns including the hydrophilicity, surface probability, chain flexibility and secondary structure Together they give rise to the antigenic index [6,7]. All peptides were extended with an extra cysteine at the carboxyl terminus to facilitate chemical coupling to the carrier protein. Coupling with m-maleimido-benzoyl-Nof the peptides to keyhole limpet haemocyanin (KLH) hydroxysuccinimide-ester (MBS) was carried out according to the method described by Kitagawa et al., [S] with some modifications [2]. Coupling to bovine sera albumin (BSA) with the use of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was performed as described by Boersma et al., [9]. The peptides were synthesized, according to the amino-acid sequence deduced from the nucleotide sequence of the A. niger bphA gene [Van Gorcom, et al., unpublished results]. The synthesis was performed on polystyrene resin (1% crosslinking) with tertiary-butyl-oxycarbonyl amino-acids, following the solid phase method as described by Merrifield [lo]. After hydrogen fluoride cleavage [ll] of the completed peptide the amino acid compositions were determined by HPLC analysis according to the method of Janssen et al. [12]. Anti-peptide antisera. Female rabbits were immunized intradermally three times with the conjugate with a monthly interval. The initial injections administered 250 pg peptide-carrier conjugate emulsified in Freund’s complete adjuvant. The conjugate for the second booster injection was emulsified in Freund’s incomplete adjuvant. Fourteen days after the second booster injection the sera were collected by bleeding of the ear vein. The sera were screened for anti-peptide reactivity by a direct and an inhibition enzyme-linked-immunosorbent assay (ELISA). Direct and inhibition ELBA. The ELISA was carried out as previously described by Boersma et al. [13]. In the inhibition assays, which were performed at the same conditions as the direct ELISA, the peptide-induced antisera dilutions and control sera were preincubated for one hour at 25°C with antigen before they were added to the wells. Westernblot analysis. Western blot analysis with the peptide antisera was performed on extracts of mycelia of two AspergiIlusniger transformants, both having multiple copies of the bphA gene. The assay was carried out according to a standard procedure [14] with fractions of cellextracts of benzoate induced and non induced Aspergihs n&r strains Results

Peptide selection and amino acid composition. Depicted representation indexrevealed [Table to the

and

of the data concerning secondary

three

structure

sequences

with

l] were synthesized carrier-protein.

The

the hydrophilicity-,

predictions

of the BPH

a consecutive

with an additional amino

acid

surface

congenial

in

figure

1 is the

graphical

probability-,

flexibility-,

antigenic

protein.

(positive)

Examination antigenic

cysteine at the carboxyl

composition 34

of the

terminus

peptides

of those

index. These

was

data,

peptides

to enable coupling confirmed

by acid

Vol.

167,

Log Surface

NID.

1, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Prob.

Flexibility 0.8

Antigenic

Index ‘...>.i:. ‘.‘.‘.: f

-1.7

CF Alpha

Helices

CF Beta

Sheets

GOR Alpha

IV

Helices

GOR Beta

Sheets

Glycosyl.

nn n

m

Sites I I I I1 b ’

..:..n

95%

followed

by HPLC

and 75’% respectively.

analysis.

structure selected

prediction data for the sequences with a consecutive

Purity of peptides

In contrast

dissolve in 5% acetic acid and no optimal

after three

all the three

peptides

homologous

immunizations induced

(non-conjugated)

AMINO-ACID

to peptides

SP032

conjugates

is demonstrated

antibodies

peptides

SEQUENCES OF POSITION

which

aminoacid sequence high antigen index

and SP034, peptide

peptide

in figure

2. The

reacted

conjugates

recognized

AND

THEIR

SPO33

NH2-Phe-Val-Pro-Glu-Arg-Trp-Asp-Pro-AlaArg-Leu-Thr-Pro-Arg-Gln-Lys-Cys-COOH

433-448

SPO34

NH2-Asn-Thr-Arg-Asp-Arg-Ala-Glu-His-ThrArg-Lys-Arg-Lys-Thr-Val-Cys-COOH

115-129

of

the respective

also with these peptides

TABLE 1 THE SYNTHETIC PEPTIDES WITHIN THE BPH MOLECULE

35

of anti-peptide

KLH-MBS

residue 199-21s

code

SP033 did not

the production

not only specifically

but in addition

of are

were obtained.

seauence NH2-Leu-Asp-Lys-Gly-Lys-Asp-Phe-Ala-GluMet-Arg-Lys-Thr-Pro-Asp-Ser-Pro-Cys-COOH

pentide SP032

l-l

SP032, SP034 and SP033 exceeded 95%,

Direct and inhibition ELZSA. For each synthetic antibodies

.‘.:.:.: .. ::::::::::. ::.::.

II-

.::.

I 1 // I I I, I I I I I I I I I f I I I I I I I I I, I I I I I I I I I, I I I I I I I I I, I ml a-m 300 400 SO0 spo34 ~po32 wN3

Figure 1. A graphical representation of the benzoate-para-hydroxylase. The three depicted in the shaded areas.

hydrolysis

ULJ-I

conjugated

RESIDUE

no

Vol.

167,

No.

BIOCHEMICAL

1, 1990

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1.50

0.60

0.30

0.00 free

BSA-edc-peptide

peptide

BSA-edc-ap0

13

coating The antibodies in the serum of rabbits immunized with KLH-MBSSPO32 MBS-SP033 (crossed area’s) and KLH-MBS-SP034 (hatched area’s) were on a coating of the peptides the peptide conjugates and an irrelevant EDC-SP013). The results of 150 sera dilutions are shown.

with an alternative coupling method (EDC)

(black area’s), KLHtested in a direct ELISA peptide conjugate (BSA-

to another carrier (BSA). No response was measured

when the sera were tested on a coating of an irrelevant peptide conjugate (BSA-EDC-SP013). The highest response was observed with peptides SPO32 and SP034. In the inhibition assays it was shown that the anti-peptide antiserum response could be inhibited with the specific peptides or specific peptide conjugates. Preincubation with peptides and peptide-conjugates not related to BPH did not inhibit the anti-peptide

response et al. [Fig. 31. Similar results were

obtained with

inhibition assays with the anti-peptide SP033 and SP034 sera respectively (results not shown).

1.50

r

125

s $ ‘Do s

0

0.75

P 0.50

.t 1

i 0.25 0.00

2 4 a 25!L

i

032

B-e-032

K-032

preine&ation Figure

B-e-01 with

3

013

anti-

3. of a KLH-MBS-SP032 immunized rabbit, after 1 hour preincubation with increasing amounts of specific antigens SP032, BSA-EDCSP032, KLH-MBS-SP032 or irrelevant antigens BSAEDCSP013 and SP013, was tested in an inhibition assay on directly coated SP032. Indicated is the amount of potential inhibitor added. The results of 150 sera dilutions are shown.

Serum

36

Vol.

167,

N’o.

1, 1990

strain: induced: marker: fraction:

Figure 4. Comparison of Pl=membrane induced strains weight marker

BIOCHEMICAL

T16

AND

BIOPHYSICAL

T21

RESEARCH

T16 +

COMMUNICATIONS

T21 +

RM PO Pl

Sl

PO Pl

Sl

PO Pl

Sl

PO Pl

Sl

the reactivity of the anti-SP032 antiserum on mycelia extracts (PO=total fraction, fraction, Sl=soluble protein fraction) of benzoate induced strains with the non of transformed A. n&r in a Western blot. The middle lane contains a molecular (RM=rainbow marker).

strain: induced: marker: fraction:

T16

T21

T16 +

T21 +

RM PO Pl

Sl

PO Pl

Sl

PO Pl

Sl

PO Pl

Sl

Figure 5. Comparison of the reactivity of the anti-SP034 antisera on mycelia extracts (PO=total fraction, Pl=membrane fraction, Sl=soluble protein fraction) of benzoate induced strains with the non induced strains of transformed A. n&r in a Western blot. The middle lane contains a molecular weight marker (RM=rainbow marker). Double staining was performed with anti-SP032 and SP034 antisera respectively.

37

Vol.

167,

No.

IV&em

1, 1990

BIOCHEMICAL

bloc analysis.

fraction

(Sl)

of extracts

reacted

very specific with

Western

blot. The

41. The

anti-SP034

(results

protein.

The

anti-SP032

the anti-SP032

recognized

A double

anti-SP034

sera; only one band (mw=46

also a benzoate

extracts induced

staining

with

serum and the additional

(Pl)

and the soluble

of BPH with

benzoate

kd) was observed

of the non-induced protein

on the

strains [Fig.

but in addition

which were not induced

the anti-

by benzoate

anti SPO32- and anti SP034 serum respectively

anti SP032

serum detected

COMMUNICATIONS

fraction

to expression

with one or more proteins

that both

RESEARCH

(PO), the membrane

serum did not react with

crossreactivity

to confirm

BIOPHYSICAL

of mycelia of both strains induced

serum

not shown).

performed

The total extract

anti-SP032

SP034 sera showed

AND

and SP034

antisera

the same (induced)

proteins

which

were

recognized

protein

the same induced

band as was detected

not induced

was

by benzoate

by the

[Fig. 51.

Discussion This

report

describes

specific polyclonal

the successful

antisera for immunodetection

sequence

as starting

difficulties

concerning

point,

this approach

the isolation

This study demonstrates derived from separate of peptides peptides their

SP032,

SP033

anti-peptide

comparable

with

the anti-KLH

response

anti-KLH

of the anti-SP033

conjugates,

during

the conjugate

expected

After

formation.

screening

blot. The

of the benzoate

kd deduced integral

from

membrane

results also showed might explain

the DNA proteins

protein

sequence often

For this purpose

antisera

met in attempts

pan

fractions

Therefore,

with

was

the low

immunization ratio. Peptide-

if the peptide

is soluble

and therefore

it was

formed. antisera

of extracts of two different

one protein

induced

in a

band (mw=46

strains.

The molecular

to the expected molecular weight of 58 Gorcom

behaviour

was associated

et al., unpublished on SDS-polyacryl with

to purify the BPH 38

in ELISA

the peptide-specific

of both benzoate

show a aberrant

to inhibit

the anti SP032 and SP034 antisera

specifically

was not equal analysis

conjugates

by an inefficient

and peptide-conjugate,

fractions

were

respective

antisera.

ratio were

tested with

which

specificity.

SP033 was not easily dissolvable

that the bulk of the BPH

the difficulties

and peptide

two peptide

a low peptidelcarrier

extract

induced

the

on their

is due to a low peptide/carrier

SP032 serum reacted

kd) in blots of all the mycelia

peptides

responses

ratio, can only be obtained

of native protein.

anti-peptide

specific

sera can not be explained

strains of Aspergihs niger were

circumventing

The antisera raised with conjugates

the above mentioned

of the other

Peptide with

mono-

With only the DNA

three

of the KLH-MBS-SP033

of the sera on peptide

were used for detection transformed

weight

confirmed

with a high peptide/carrier

that only conjugates

to generate

of BPH,

raised against

of specific peptides

It is more likely that the low response

carrier

detection

in ELISA

response

response

peptides

of the native molecule.

of antibodies

The ability responses

of the

Western

and purification

and SP034 showed

level

procedure.

specific

regions of the benzoate-para-hydroxylase.

The

anti-peptide

of synthetic

of benzoate-para-hydroxylase. allows

properties

and peptide-conjugates.

respective

application

the membrane

protein

results],

amide

but

gels. The

fraction.

‘Ihis

from mycelia. I’he anti-

Vol.

167,

No.

1, 1990

BIOCHEMICAL

SP032 sera did not eact with with

anti-SP0.32

respectively

and double

control

extract

staining

recognize

or conformational

are also present

similarity

represented

by the synthesized

sequencing

of these non-identified

of the 115-129

proteins

This study shows that prediction DNA

sequences

specific

reagents

synthetic opens

peptides

can be a useful for detection

new possibilities

in functional

and/or

mono-specific

fractions

and one

of the

amino acid sequence

confirm

to the practical

of proteins

and corresponding

could

strains.

Single

staining

and anti-SP034 However

sera

the anti-SP034

but additional

proteins

strains. Possibly, there is a sequencepossible

conformations

of BPH. Isolation

and

this hypothesis.

and synthesis of putative

tool

protein.

in the induced-strain

proteins

COMMUNICATIONS

antiSP032

the same induced

these

peptide

blot with

in the non induced

between

RESEARCH

samples of the non-induced

not only the BPH protein

as well. These proteins

BIOPHYSICAL

of the Western

shows that both antisera

antisera recognize

AND

antigenic

biochemist

protein

fragments.

polyclonal

studies and purification

determinants

using only

in the production

of highly

The

availability

of selected

sera specific for the native sequence of the BPH

enzyme.

Acknowledgments We composition Geleen,

The

thank

Dr.

J.M.

van

of the synthesized

Noort peptides.

and This

Mrs.

J. Boon

for

study was supported

determination in part

the

amino-acid

by DSM

Research,

Netherlands.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Fearny, F.J., C.Y. Leung, J.D. Young and E. Benjamini. (1971) Biochem. Biophys. Acta. 243, 509-514. Van Denderen, J., A Hermans, T. Meeuwsen, C. Troelstra, N. Zegers, W. Boersma, G. Grosveld and W. van Ewijk. (1989) J. Exp. Med. 169, 87-97. Boersma, W.J.A., C. Deen, K. Gerritse, N.D. Zegers, J.J. Haaijman, and E. Claassen. (1989a) Prot. Biol. Fluids, in press. Boersma, W.J.A., J.E.M. Van Leeuwen, C. Deen, J. Radl, J.J. Haaijman and E. Claassen. (1989b) Prot. Biol. Fluids, in press. Francis, M.J., G.Z. Hastings, A.D. Syred, B. McGinn, F. Brown and D.J. Rowlands. (1987) Nature 330, 168-170. Wolf, H., S. Modrow, M. Motz, B.A. Jameson, G. Hermann and B. Mrtsch. (1988) Cabios 4, (l), 187-191. Jameson, B.A and H. Wolf. (1988) Cabios 4 (1) 181-186. Kitagawa, T. and T. Aikawa. (1976) Biochem. 79, 233-236. Boersma, W.J.A., E. Claassen, C. Deen, K. Gerritse, J.J. Haaijman and N.D. Zegers. (1988) Analytica Chimica Acta 213, 187-197. Merrifield, R.B. (1963) J. Am. Chem. Sot. 85, 2149. Bhatnagar, PK., S.J.T. Mao, A.M. Gotto, Jr and J.T. Sparrow. (1983) Peptides 4, 343-349. Melgers, H.W.M. van den Bogaart, R.L.A.E. Janssen, P.S.L., J.W. van Nispen, P.A.T.A. Hamelinck and B.C. Goverde. (1986) Chromatographia 22, 351-357. Boersma, W.J.k, E. Claassen, C. Deen, K Gerritse, J.J. Haaiman and N.D. Zegers. (1988) Anal. Chim. Acta, 213, 187-197. Towbin, .H., T. Staehelin and J. Goordon. (1979) Proc. Natl. Acad. Sci. 76, 4350-4354.

39