Versatility of a vector for expressing foreign polypeptides at the surface of Gram-negative bacteria

181

Gene, 70 (1988) 181-189 Elsevier GEN 02622

Versatility of a vector for expressing foreign poiypeptides at the surface of Grab-negative bacteria (Recombinant DNA; maltoporin; outer-membrane)

phage A; protein engineering; secretion; vaccines; expression vectors;

Alain Cfaarbit, Annie Molla, William Saurin and Maurice ~ofnung CNRS-UA271, INSEM-U163, Received

29 March

Accepted

30 May 1988

Received

by publisher

Unit4de ProgrammationMokculaire et Toxicologic G&&ique, InstitutPasteur 75015 (France)

1988 5 July 1988

-

SUMMARY

A wide variety of peptides in terms of length and sequence can be expressed at the surface of the bacterium by genetic insertion into a ‘permissive’ site of the outer membrane protein LamB, used as a carrier. The resulting hybrid proteins essentially keep their biological activities with inserts of up to about 60 amino acid residues, and of a large range of predicted structures or hydrophobicities. This reflects a remarkable flexibility in the organization of the protein, but also in the export machinery. The method used to select such a permissive site is quite general and its potent&J to generate app~cations, including a versatile type of live bacterial vaccine, are discussed. Escherichia coli

The outer membrane of Gram-negative bacteria plays an essential role in the interaction with other biological entities such as antibodies, bacteriophages, other bacteria, or higher cells (Nikaido and Vaara, 1985). These ~teractions are mediated thanks to specific moiecular motifs located at the cell surface. We have recently been able to modify genetically the surface of the bacterium E. coli by exposing foreign antigenic determinants. This was done by

genetic insertion into the outer membrane protein LamB, used as a carrier (Char-bit et al., 1986; 1987). LarnB is located in the outer membrane of E. co& where it exhibits two biological activities: (i) it is involved in the entry of maltose and maltodextrins into the cell (hence the name: maltoporin) {Szmelcm~ and Hofnung, 1975) and (ii) it serves as surface receptor for several bacteriophages, including bacteriophage 1 (hence the alternative name; I receptor) (Thirion and Hofnung, 1972; RandallHazelbauer and Schwartz, 1973; Charbit and

Corre~~Qnde~ce to: Dr. M. Hofnung,

hepatitis

INTRODUCTION

UPMTG,

25 rue du Dr. Roux, 75015 Paris (France)

Institut

Pasteur,

Tel. (1)4558 8830.

HIV,

B virus; hi, hydrophobic

human

immunodeficiency

toxin (see Table Abbreviations: enzyme-linked

aa,

amino

immunosorbent

disease virus; FRHD,

acid(s); assay;

Ap,

ampicillin;

FMDV,

fusion related hydrophobic

ELISA,

foot and mouth domain;

HBV,

II); IPTG,

side; oligo, oligodeoxynucleotide; line;

SDS,

DUCTION

0378-l 119/8S/$a3.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

sodium

dodecyl

and Fig. 1.

index (see legend to Table II); virus;

isopropyl

HS

toxin,

heat-stable

/.?-D-thiogalactopyrano-

PBS, phosphate-buffered sulfate;

site

sa-

153, see INTRO-

182

Hofnung,

1985). The active form is a trimer.

The

protein is synthesized as a precursor with an N-terminal signal peptide which is cleaved upon export to yield monomers of 421 aa residues. Upon induction by maltose or maltodextrins, LamB becomes a major component Schwartz,

of the 1987).

outer

membrane

(review

in

We had previously inserted an artificial BamHI restriction site between aa residues 153 and 154 (named

site 153) (Boulain

et al., 1986), in a loop

notice that, for each double-stranded oligo, in the coding orientation the 5’ end was a BamHI half site while the 3’ end was a BgZII half site. Thus upon insertion in the correct orientation, a single BamHI site was restored at the 5’ end of the insert, and not at the 3’ end. This was convenient for two reasons. On the one hand, it provided a restriction analysis screen for insertions that occurred in the correct orientation. On the other hand, it permitted successive tandem insertions of oligos by using the single

facing the cell exterior according to our folding model

restored

(Gehring synthetic

LamB-AB was double-stranded

et al., 1987; Charbit et al., 1988a). When a oligo, encoding 13 aa residues and includ-

ing the C3 epitope from poliovirus (Horaud et al., 1987), was cloned at this site, this epitope was accessible to specific monoclonal antibodies at the bacterial surface (Charbit et al., 1986). The hybrid protein was stable, could be produced in large amounts without toxic effects and presented the two biological activities of LamB. All this indicated that its folding and location was similar to that of LamB and was a direct confirmation for the cell surface location of the region of site 153, as proposed in our model. In the present paper, we study the degree of permissiveness of site 153. We show that polypeptides of a wide range of sizes and of sequences can be expressed by the same procedure, leading to an artificial form of antigenic variation.

MATERIALS

AND METHODS

(a) Strains, oligodeoxynucleotide and chemicals

insertions,

media

Strain pop6510 (thr Zeu tonB ZacYl recA dex-5 metA supE) was used as recipient for all transformations. Plasmid pAJC264 was used for all oligo insertions into the IamB gene. It was derived from plasmid pAC1 by insertion of a BamHI linker between aa residues 153 and 154 of the LamB protein (Fig. 1); plasmid pAC1 carries gene IumB under control of the ptucl2 promoter as well as a copy of the luclQ gene (Boulain et al., 1986). Single-stranded oligos encoding the various peptide sequences (see Tables II and III) were synthesized by J. Igolen (Institut Pasteur, Paris) together with the complementary strand. It is important to

BamHI

site. Thus,

for example,

obtained after oligo encoding

hybrid

insertion of the peptide preSZA

into the single BamHI site of construction LamB-B, located 5’ to preS2-B. Media and chemicals were as in Boulain et al. (1986). (b) Characterization

of the hybrid LamB proteins

All the constructions were checked first at the DNA level by restriction analysis (not shown). The hybrid proteins were then examined by immunoblotting of crude bacterial extracts (Charbit et al., 1986), using a polyclonal anti-LamB serum (see Figs. 2 and 3). Induction of LamB expression was performed by addition of IPTG (lo- 3M final). Bacterial extracts were heat-denatured (100” C for 10 min) and loaded on an SDS-polyacrylamide slab gel (Boulain et al., 1986). In vivo phage receptor activity of the different hybrids was assayed by cross-streak on ML medium plates supplemented with 100 pg Ap/ml against a wild-type host range i phage (noted Ah+), and a phage mutant (Ahh*) with the most extended host range (Hofnung et al., 1976).

RESULTS

AND DISCUSSION

We will explore successively the limit in length and nature for inserts at site 153, and consider some implications of the results. (a) Maximal

length of inserts

To determine the maximal acceptable length, we constructed tandem insertions of increasing sizes.

183

For this, we chose two peptides of the PreS2 region from the envelope protein from HBV. We had already shown that, similar to the C3 epitope, each of these peptides could be expressed at site 153 in the form of hybrid proteins LamB-A and LamB-B (Charbit et al., 1987). Peptide A was 17 aa residues long and very hydroph~ic; peptide B was 20 aa long and rather hydrophobic (Tables I and II). We started from LamB-A and LamB-B to generate LamB-AB and LamB-BA hybrids. These correspond to an insertion of 37 aa residues; (due to the initial BamHI linker, the total insertion is 41 aa residues). With respect to biological activities, LamB-AB behaved as LamB-A and LamB-BA as LamB-B (Table I; Charbit et al., 1987). The two hybrid proteins were detected in normal amounts by

immunoblotting of crude bacterial extracts under denaturing conditions with a polyclonal anti-LamB serum (Fig. 2). On intact cells, as determined by ELISA, LamB-AB and LamB-BA were recognized by the anti-LamB serum and by a polyclonal antiPreS2 serum. This confirmed that the two hybrid proteins were at the cell surface and indicated that the PreSZA epitope was facing the exterior. We then inserted tandem repetitions of peptides A and B, respectively. With peptide A, recombinant plasmids with up to five repetitions could be recovered (LamBAAAAAB abbreviated as A5B). The amount of hybrid protein detected by immunoblotting for A2B (insert of 54 aa residues) was still normal, but it was decreased for A3B (71 aa residues), more for A4B

Vector

TCC CGG AGG GCC CTA G ser arg asp 153

GAT CCC GCC TCT GG CGG AGA asp pro ala ser 154

fnserts

PreSZA 145

132

S'GAT

pro cc;G Gc

gin asp pro arg val arg gly

leu tyr phe pro ala gly gly leu

CAA GAT CCG CGT GTT AGA GGT CTC TAC TTC CCG GCA GGT GGT CTA GTT CTA GCC GCA CAA TCT CCA GAG ATG AAG GGC CGA CCA CCA GAT

CTA G 5'

PreS2B 153 Pro

5'GAT

ct;% &c

171 val

ala

leu thr thr

Leu ser ser ile phe ser arg ile gly asp

GTT CTC ACC ACC GCT AGC CCG CTC TCC TCC AX

and PreS2-B peptides

of site 153 in LamB, as well as the nucleotide The corresponding

amino acid sequences

for the two inserts,

PreS2-A

LamB at positions

153 and 154 are indicated

protein).

The aa residues

with the BamHI

are printed

above the nucleotide

sequences

are indicated

and PreS2-B,

orientation

pro

TTC TCC CGT ATC GGT GAT CCA

from HBV. The figure shows the nucleotide of the inserts.

above the nucleotide

corresponding

sequence

by the BarnHI

in lower-case-letters.

with aa residue

from aa 153 to 171 (see also Table II).

120. PreS2-A

sequence

corresponding

to the restriction

for LamB,

CTA G 5’ to the region

sites are underlined.

and under the nucleotide

sequence

site is shown as a large gap. The two serine residues

in normal lettering below the nucleotide

encoded

sequence

Sequences

from HBV. Vector. The BamHI

sequence

(numbering

from

starts at the first aa residue

linker are in italics. Inserts. The PreS2 inserts are shown in the correct

half site in 5’ position on the transcribed

the virus. The PreS2 region starts extends

pro

CAA GAG TGG TGG CGA TCG GGC GAG AGG AGG TAG AAG AGG GCA TAG CCA CAT GGT

Fig. 1. Site 153 and the PreS2-A

of the mature

ser

strand

and the BglII half site in 3’ position.

They are numbered extends

Residues

from PreSt

from the first aa residue in the PreS2 sequence

from aa 132 to I45 in the HBV PreSZ-A

sequence.

of

PreS2-B

184

(88 aa residues),

and was undetectable

in A5B (insert

obtained after insertion of an additional B sequence, suggesting that hybrid B3A was deleterious to the plasmid or to the cell. [It should be recalled that the basal level of expression of the plasmid-carried LamB gene is significant even in absence of inducer

of 105 aa residues). In vivo phage receptor activity was decreased but detectable, for A2B ; it was undetectable for A3B and A4B. Hybrid protein A2B was detected at the surface of intact cells by the antiLamB and anti-PreS2 antibodies, while A3B and

(Boulain et al., 1986).] Phage receptor activity was slightly affected, but detectable, in hybrids LamB-B,

A4B were not detected. With peptide B, LamB-B2A mal amounts

serum (Fig. 2). However,

TABLE

in nor-

BA and B2A. Hybrids

with the anti-LamB

no transformants

could be

BA and B2A were detected

by ELISA

at the surface of intact cells, by both the

anti-LamB

and anti-PreS2

antibodies

(Table I).

I

LamB-hepatitis Inserts

was detected

by immunoblotting

B peptides

coded by genetic Number

at site

hybrids a

of

ahh* d

Denatured

residues c

LamB153b

extracts

Intact

immunoblot anti-LamB

cells

ELISAf e anti-LamB

anti-PreS2 (anti-A)

(BanzHI

site)

(4)

S

++

+

_

A

11

S

++

+

+

AB

31

S

++

+

+

A2B

54

RS

++

A3B

71

R

+ _

A4B

88

R

-

_

A5B

105

R

+I& _

+ -

_

B

20

RS

++

+

-

BA

31

RS

++

+

+

B2A

57

RS

++

+

+

(77)

(B3A) a Double-stranded

synthetic

oligos corresponding

site (site 153) of the modified

lamB gene carried

residues:

by BamHI

R-D-P-A.

Cleavage

b Nature

of the insert. AnB means

c Length

of the insert (in aa) exclusive

d Sensitivity

All these hybrid proteins

n copies of peptide by cross-streak

mutant

to wild-type

(Fig. 2); -, no protein detected. f ELISA with intact bacteria microtiter (100 pl of bacterial

for anti-LamB

and anti-PreS2

The polyclonal

at room temperature. anti-PreS2

strain ~0~6510, was at least 2 : 1; symbol

AND METHODS,

antibodies.

+ + wild-type

amounts;

by overnight

Reactions

incubation

at 37°C

was continued

were revealed

LamB antiserum

+ indicates

- indicates

section a) is the LamB-negative

+ / - reduced

amounts; with approx.

of l/4 000, l/4 000, l/l500

reacted

with HBV surface antigen recombinant

Symbol

with 100 pg

RS, less sensitive than wild type; R, resistant.

E trace amounts 5 x lo6 intact

were used, respectively,

with 0.5 % gelatin in the same buffer, 1 h at 37°C. Then, 100 ~1 of dilutions

The polyclonal

serum reacted

region A, but not region B, in the LamB hybrids control

serum.

plates (Nunc Inc.) were coated

anti-rabbit

B at site 153.

In the test, a single insert B, but not a single insert

0.5% gelatin) were added per well, and incubation

100 ~1 of peroxidase-labelled

sulfonic acid) as substrate, oligomers.

host range phage I (ah+).

sera. Wells were post-coated

4 aa

et al., 1986).

plates (see Boulain et al., 1986) supplemented

dilution at A 6oo = 0.1 in PBS buffer) per well. Dilutions

of serum (in PBS buffer containing wells received

by one copy of peptide

(Boulain

into the BarnHI

of a linker encoding

linker.

et al., 1976). S, fully sensitive;

with anti-LamB

to the addition

the R and D residues

on ML medium

of A (Hofnung

confer resistance

This site corresponds

is between

A followed

of the initial BamHI

A, slightly affected phage adsorption. e Detection of hybrid protein by immunoblot

bacteria

A and B, from the PreS2 region of HBV were inserted pAJC264.

on the coding strand

to phage Ihh* as determined

Ap/ml. Ihh* is a broad-host-range

to peptides by plasmid

for 2 h at 37°C. After washing,

with 2-2’-azino-di-(3-ethyl-benzthiazolin with LamB monomers, particles,

that the signal ratio between

the mutant

and higher

strain and the negative

that the signal ratio was less than 2 : 1. Strain pop6510

strain used as a recipient

trimers

the PreS2 region of the HBV, and

for all the constructions

(Charbit

(see MATERIALS et al., 1986).

18.5

TABLE II Hydrophobicity of inserted sequences Designation in text a

Amino acid sequence b

Inserted aa residues c

Hydrophobicity d Total

Local

Origine

(A) (B) (1) (2) (5) (8) (9)

17 20 25 17 24 13 44

-0.01 -0.14 + 0.27 + 0.21 +O.lO -0.46 -0.22

-0.98 + 0.43 + 0.68 + 0.72 + 0.96 -0.6 -0.76

HBV HBV HIV1 HIV1 HIV1 Poliovirus FMDV

(6)

24

-0.64

-1.28

HIV1

+ 0.02

+ 0.82 -0.89

E. coii

(11)

LamB preprotein (446 aa residues)

a Name attributed to the sequence in Figs. 2 and 3, and in the text. b Amino acid sequences: bold-face capital letters correspond to the aaresidues ofthe foreign organism and numbers refer to the position of the first residue. References for sequences: HBV (Michel et al., 1984); HIVI, (Alizon et al., 1986); poliovirus (Horaud et al,, 1987); FMDV (Bittle et al., 1982); HS toxin (Thompson and Giannella, 1985); LamB (Clement and Homung, 1981); small capitals correspond to extra residues encoded by the extremities of the synthetic oligo. The FMDV insert was assembled from two tandem sequences which are not contiguous in the virus. c Length of the inserted sequence in amino acid residues and except the initial BumHI linker. d Hydrophobicity of foreign sequences:,values were calculated over 9 aa long segments with a normalized hydrophobicity scale (Eisenberg et al., 1984). The total hydrophobic index of a foreign sequence is defined as an average of its hydrophobicity values, Local hydrophobic index is defined as its highest ( t ) or lowest ( -) hydrophobicity values. e Organism from which the inserted sequence was issued.

In summary, two different sequences of respectively 54 aa residues (A2B) and 57 aa residues (B2A) led to the detection of the PreS2A epitope on bacterial cells by ELISA. Constructions with larger inserts led either to the detection of decreasing amounts of the proteins, presumably due to decreasing stability (from A3B to A4B and A5B) or to non-recovery of tr~sfo~~ts (B3A), which we attributed to toxicity of the hybrid proteins. Other explanations such as effects of the repetitive sequences on gene expression (for A3B, A4B and A5B) or on plasmid replication (for B3A) cannot be excluded but appear less likely. At any rate, the size limit appears to be between 60 and 70 aa residues and depends on the exact nature of the sequence. This size could possibly be increased by modifying the genetic background of the strain. For example, some hybrid proteins may prove less unstable in bacterial mutants affected in certain proteases

(Strauch therein).

and

Beckwith,

1988, and

references

123456789 Fig. 2. Lamb-PreS2 hybrid proteins. The hybrid proteins correspond to tandem insertions of PreS2-A and PreS2-B at site 153 from LamB. Heat-denatured bacterial extracts of the different strains (MATERIALS AND METHODS, section b) were loaded on SOS-lO~O polyacrylamide slab gel. After electrophoretical transfer onto a nitrocellulose filter, hybrid proteins were visualized by using a polyclonal anti-LamB serum at a final dilution l/500. Lanes: 1, LamB-B2A; 2, LamB-BA; 3, B; 4, A5B; 5, A4B; 6, A3B; 7, LamB-A2B; 8, LamB-AB; 9, A.

186

(b) Inserts of different nature

MalE, of E. coli remained attached to the outer face of the inner membrane when signal peptide remained

Out of over 25 peptides that we have now expressed in site 153 of LamB, we selected twelve which represent a wide spectrum of hydrophobicity

uncleaved (Dalbey and Wickner, 1985; Fikes and Bassford, 1987). The hi’s of these signal peptides were: 0.63 in the case of MalE with 18 consecutive

(Table II) and predicted

uncharged aa residues, and 0.90 in the case of OmpA with 20 consecutive uncharged aa residues. The fact

FMDV,

structure

(Table III). They

from very different sources : HBV, HIVl,

originated

poliovirus

that sequence

and HS toxin. The hybrid pro-

protein

teins were all functional for adsorption of phages using the LamB protein and were present in normal amounts

as judged by immunoblotting

terial extracts

with an anti-LamB

suggested that a string of, at least, 16 to 17 uncharged aa residues was needed for membrane anchoring of

serum (Fig. 3).

(I) Hydrophobicity Three sequences (8, 9, 6) were hydrophilic, with hydrophobic indices ranging from -0.22 to -0.64. The region of insertion in LamB around site 153, is hydrophilic (local hi = -0.30). Sequence (6) from HIV1 presented the highest local hydrophilicity (local hi = -1.28). It was more hydrophilic than the most hydrophilic peptide in LamB (local hi = -0.89). Three sequences (1, 2, 5) were hydrophobic, with hydrophobic indices from 0.1 to 0.27. Sequence (5) from HIV1 presented the highest local hydrophobicity (local hi = + 0.96). This was higher than that of the LamB signal peptide (local hi = + 0.82), the most hydrophobic sequence of the IamB product. It has been shown that uncleaved signal peptides can behave as inner-membrane anchors for exported proteins. For example, the outer membrane protein

presented

OmpA and the periplasmic

each table).

TABLE

could be due to the

relative short length of the uncharged sequence (14 consecutive uncharged aa residues). Indeed, it was

of crude bac-

maltose-binding

(5) does not block the LamB hybrid

in the inner membrane

a normally secreted form of protein pII1 from coliphage fl (Davis and Model, 1985). We cannot exclude that hydrophobic sequences with a higher local hydrophobicity than sequence (5) could prevent export of the LamB hybrid. Indeed, it was demonstrated that the hydrophobic anchoring domain (HAD) of the Sendai virus F protein (hi = 1.23,

1 2

3

Fig. 3. LamB hybrid proteins were visualized extracts

protein,

5

indicated,

6

7

8 91011

with other inserts. Hybrid proteins

by immunoblotting

with a polyclonal

2). The numbers

L

of heat-denatured

bacterial

anti-LamB

serum (see legend of Fig.

correspond

to the different sequences

in Tables II and III (see column a with footnote

a, in

III

Secondary Designation

structure

predictions Amino

for inserted

peptides

acid sequence’

in text a

Inserted

Coil + turn

Structured

aa residues’

in y0 d

in %’

Origin f

(8)

DP=DNPASTTNKDK

13

100

0

poliovirus

(10)

DP’NTPYCCELCCNPACAGCYVK

22

80

20

HS toxin

(3)

DP~~EIVTHSFNCGGEFFYCNSTQLFNS

28

17

23

HIV1

(4)

DP~~ELYKYKWKIEPLGVAPTKAKR

24

9

86

HIV1

(5)

DP~=RWQREKRAVGIGALFLGFLGA

24

23

II

HIV1

(7)

DP=‘RILAVERYLKDQQLLGIWGCS

23

29

71

HIV1

a~b,c,f See legend to analogous d % of the polypeptide e % of the polypeptide predicted

columns

chain predicted chain predicted

as a coil or turn (Garnier

in Table II. as a coil or turn structure. as structured;

et al., 1978).

we defined structured

regions as segments

longer than 3 aa, none of which being

187

higher than that of sequence (5), but not another hydrophobic domain of the same protein [the fusionrelated domain (FRHD), hi = 0.87, lower than that of sequence (5)] could anchor the normally secreted form of protein pII1 (Davis and Hsu, 1986). However, in these two cases, the continuous strings of uncharged aa residues were also longer than in sequence (5) (respectively, 24 and 26 aa residues). (2) Other aspects The amino acid composition of the inserted peptides can be very peculiar: for example, peptide (10) included two proline and six cysteine residues. The corresponding hybrid LamB protein was stable, exported and active. The predicted structures can also cover a wide range. Three sequences (8, 10, 3) were predicted as coils or turns forming regions with a value of 100% for peptide (8): this peptide corresponds to a turn in the polio~us VP1 protein (Horaud et al., 1987). Three sequences (4, 5, 7) exhibited from 71% to 86% of predicted structured region (a-helix or /?-sheet) (Table III). (c) Permissivity

of site 153

In conclusion, site 153 shows a striking permissivity, in terms of the length and nature of the peptides which can be expressed without affecting in any impo~~t way the mount, activities or localization of the hybrid protein. This reflects a remarkable flexibility in the organization of the protein but also in the export machinery. Recently, an 8-aa peptide from the C-terminal part of FMDV was inserted after aa residue 158 of PhoE, another trimeric outer membrane protein from E. coli, and exposed at the cell surface (Agterberg et al., 1987). It was also suggested that OmpA, a monomeric outer membr~e protein, might be valuable for the same purpose, since insertion of 15 foreign residues between aa 153 and 154 made the protein sensitive to proteolysis from outside the cell (Freud1 et al., 1986). In both cases, a restriction site had been created in a region presumed to be exposed at the cell surface. In the case of gene lumB, site 153 was experimentally determined. Two steps were used. First, a BamHI Sinker was inserted at random into gene IamB and we screened for stable modified proteins

(Boulain et al., 1986). Second, we examined which of the newly created sites could accommodate further insertion corresponding to the C3 epitope (Charbit et al., 1986). We have since used this procedure to detect other permissive sites in LamB, and in other proteins (Duplay et al., 1987; P. Duplay, P. Martineau and MI-I., unpublished data). It thus appears quite general. The degree of permissivity depends on the site and on the protein. One open question is : to which features of the protein do such sites correspond? One possibi~ty is the recently described omega loop structure (Leszcinsky and Rose, 1986). There are certainly other possibilities. Precise structural data on LamB, when available, should help elucidate this point. At any rate, the existence of such sites and the availability of an simple genetic method to detect them, even in absence of structural data, offer a powerful approach for engineering chimeric proteins. One interesting aspect of the chimaeras produced is that all, or most, of the activities of the carrier protein are conserved. Thus, topological conclusions drawn from the properties of the hybrid proteins are likely to be valid for the carrier (Charbit et al., 1986). In addition, depending on the carrier protein, this conservation has a number of potential applications. In the case of LamB, presentation of foreign antigenic determinants at the bacterial surface, may lead to elaborate multip~pose live bacterial vaccines (Charbit et al., 1988b,c). Indeed, cells of E. coli exposing the C3 epitope from polio~us elicited antibodies against the virus upon administration to experimental animals (Charbit et al., 1988~). Similarly, this mode of presentation of the PreS2-A, or the PreS2-B epitopes from the hepatitis B virus, gave rise to antibodies against the hepatitis B surface antigen particles (Charbit et al., 1987). Furthermore, when the plasmid expressing the C3 epitope was transferred into an attenuated strain of S. t)tphi, Ty2la, used for vaccination trials on humans (Ge~~ier and Levine, 1986), the epitope was detectable at the bacterial cell surface (A.C., unpublished). We have used the same kind of approach in the case of the maltose-binding protein, MalE, which can conveniently be affinity-purified. We determined permissive sites and used two of them to construct vectors for the one-step purification of polypeptides of interest (Bedouelle et al., 1987; Bedouelle and Duplay, 1988; unpubl. data from our laborato~).

188

Charbit,

ACKNOWLEDGEMENTS

A., Molla, A., Van der Werf, S., Sobczak,

M.-L., Girard,

We thank Valerie Michel for excellent technical assistance. We thank Fred Heffron (Scripps Clinic, La Jolla) for the gift of the oligos corresponding to peptides 5 and 7. We also thank David Perrin and Greetje Vos-Sherpekeuter for the gift of anti-LamB sera, and Marie-Louise Micbel for the anti-PreS2 serum. This work was supported in part by grants from the Fondation pour la Recherche Medicale, the Ligue National contre le Cancer, and the Association pour la Recherche sur le Cancer.

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of recombinant

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membrane

protein

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foreign antigenic determinant

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for the transport

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to the cell surface OfEscherichia

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A., Wain-Hobson,

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variability

virus: nucleotide

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R.E. and Wickner,

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M.: Expression,

expor-

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Sci. Paris 305 (1987)

W.: Leader

peptidase

Bittle, J.-L., Houghten, Sutcliffe,

R.-A., Alexander,

J.-G. and Lerner,

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by immunization

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with

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foot-

a chemically

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se-

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by

into the Jan& gene of E. coli K-12.

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J. Biol. Chem. 260 (1985) 15925-15931.

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