IMMUNOLOGICAL
INVESTIGATIONS, 21(3), 241-257 (1992)
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IMMUNOREACI'MTY OF RECOMBINANT CARCINOEMBRYONIC ANTIGEN PROTEINS EXPRESSED IN ESCHEZUCHIA COLJ Masahide Kuroki, Masaaki Murakami. Mamie Wakisaka. Shoichi Ikeda, Shinu, Oikawal , Takehiro Oshimal. Hiroshi Nakazatol , Goro Kosaki2 and Yuji Matsuoka First Department of Biochemistry, School of Medicine, Fukuoka University, 7-45- I Nanakuma, Jonan-ku. Fukuoka 814-01, Japan 1Suntory Institute for Biomedical Research. 1- 1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 6 18, Japan. Tokyo Metropolitan Komagome Hospital, Tokyo 170, Japan
Immunoreactivities of recombinant carcinoembryonic antigen (CEA) proteins expressed in Escherichia coli (E. coli) were analyzed in relation to the CEA domain structure [domains N, I (Al-Bl). I1 (A2-B2), I11 (A3-B3) and MI. We reconstructed in a prokaryotic expression vector, PUCPL-cI. the cDNAs for CEA-N, CEA-I. CEA-11. and CEA-111-M. The latter three were expressed as fusion products with bacterial p-galactosidase. The recombinant proteins were s o l u b i k d by sonication in 1% sodium dodecyl sulfate (SDS) and purified by preparative SDS-polyacrylamide gel electrophoresis followed by electroelution. Their molecular weights judged from Western blotting coincided with those calculated from their cDNA sequences, respectively. By solidphase enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested with 21 distinct anti-CEA monoclonal antibodies (MAbs) which had been found to recognize the peptide epitopes of the CEA molecule and to be reactive with the recombinant CEA proteins expressed in Chinese hamster ovary (CHO) cells. Fourteen of the 21 MAbs reacted with the recombinant CEA proteins expressed in E. colt and confirmed the localization of the epitopes identified by using the recombinant CEA proteins expressed in CHO cells. The reactivities of 5 MAbs with the recombinant proteins expressed in E. coli were remarkably low when compared with those of the proteins expressed in CHO cells but also confirmed the localization of the 241
Copyright 0 1992 by Marcel Dekker, Inc.
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epitopes identified with the recombinant CEA proteins expressed in CHO cells. The remaining 2 M A b s did not react with any recombinant protein expressed in E. coli. These results indicate that the fusion CEA-proteins expressed in E. coli are useful in the localization of the epitopes on the polypeptide chains when they reacted with the MAbs tested. However, one third of the epitopes of CEA peptides may be profoundly affected by the presence of disulfide bonds and/or sugar chains which do not seem to be formed well in E. coli.
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INTRODUCTION
Carcinoembryonic antigen (CEA) is a glycoprotein with a molecular weight of 180,000 and is one of the most useful human tumor markers for the post surgical monitoring of patients with various malignancies (1). In addition there is increasing interest in the in uiuo use of anti-CEA MAbs in tumor imaging (2) or therapy (3). The primary structure of CEA peptide. as determined by chemical sequence analysis (4) and successful cDNA cloning (5). has revealed that it is a member of the CEA gene family within immunoglobulin supergene family (4. 6). I t has been known that the CEA gene family consists of at least 20 different genes (7).The CEA structure is composed of the following five domains (5): a n N-terminal domain (N) of 108 residues: three very homologous repetitive domains I, 11. and 111 of 178 residues, each of which was subdivided into A and B subdomains and designated A1-B1, A2-B2, and A3-B3, respectively, by the newly proposed nomenclature (8);and a hydrophobic C-terminal domain (abbreviated as M) of 26 residues which are post-translationally replaced with a glycosylphosphatidylinositol (GPI) moiety for membrane anchoring (9.10). Furthermore, several recent works have clarified that CEA has cell adhesion activity (11.12) or bacterial binding activity (13.14). These findings suggest that it might play some important role in cell-cell interactions. Since more than 20 structurally related proteins, known to be CEArelated antigens, have been found in various human tissues or body fluids as members of the CEA gene family (7).elucidation of the specificity of MAbs used to detect CEA is very important. The specificity of anti-CEA MAbs has been evaluated by several approaches including the analysis of cross-reactivity with the CEA-related antigens (15-18). immunohistochemical reactivity with a variety of normal or malignant tissues (19-211, and competitive binding activity among MAbs (22). Recently.
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recombinant CEA proteins including fusion proteins have been produced using eukaryotic (23.24) or prokaryotic (25) expression vectors and were utilized for analyzing the specificity of anti-CEA MAbs (24.25). In recent works, we prepared several recombinant CEA- or NCA-proteins with different domain structures by expressing the corresponding genes in Chinese hamster ovary (CHO) cells, and mapped the epitopes recognized by 21 different anti-CEA (24) or 8 different anti-NCA MAbs (26) in terms of domain structure. In the present study, we expressed the fusion proteins composed of bacterial p-galactosidase and each of the 4 principal domains of CEA in E. coli, and demonstrated the utility of these proteins in mapping the epitopes of the CEA molecule. The results were compared to our previous mapping obtained with recombinant CEA-proteins expressed in CHO cells.
MATERIALS AND METHODS
Antibodies. Polyclonal anti-CEA antisera were prepared in rabbits or BALB/c mice as described previously (27.28). and specifically purified with CEA-Sepharose 4B. respectively (28). The affinity-purified BALB/c polyclonal anti-CEA antibody was used as positive control. Twenty-one different anti-CEA MAbs used in this study were prepared and purified as described elsewhere (16.18). Purified myeloma proteins MOPC 21 (IgG1) and MOPC 104E (IgM) were used as negative controls (Organon Technika, West Chester, PA). Reference CEA and NCA Preparations. Reference CEA was purified from a metastatic liver tumor from a colonic adenocarcinoma by the procedure described earlier (29). Reference NCA was purified from pooled normal human lungs by the procedures described previously (28). Construction of prokaryotic expression vectors. The expression vector used in this study was the PUCPL-CI which contains a PL promoter, cIts857, Shine-Dalgarno sequence, initiative methionine. multi-cloning sites, stop codons in any frame, and trp terminator (our unpublished vector). Restriction enzymes were purchased from Takara Shuzo (Tokyo, Japan) except for BsfX I (New England Nuclear, Boston, MA).
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PUCPL-CEA-Nwas constructed as follows. The pCEA55-2 cloned in Okayama-Berg vector (5) was digested with Hind I11 and Dra I. then excised fragment was inserted into the Hind I11 and Hfnc I1 sites of the M 13mp18 to yield a single-strand DNA template (M 13mp 18CEA55-2) for site-directed mutagenesis. The M 13mp18CEA668-8 which h a s the EcoR I site and the initiative methionine j u s t before the N-terminal amino acid, lysine, of the CEA. was produced with 33-mer oligonucleotide, 5-TGGAACCCGCCCGAA'ITCATGAAGCTCACTAlT-3'using the oligonucleotide-directed in uitro mutagenesis system (Amersham. Buckinghamshire. England) and then digested with the EcoR I and Sma I, then the excised fragment was inserted into the EcoR I and Sma I sites of the PUCPL-CIto yield PUCPL-CEA. The PUCPL-CEAwas digested with Acc I, blunt ended with T4 DNA polymerase and the large fragment was self-ligated to yield the ~UCPL-CEA-Nwhich h a s the open reading
frame of the initiative methionine followed by CEA-N-domain (106 amino acids, from Lysl to VallO6) with a n additional artificial six-amino acid sequence. STCRHA a t the C-terminus. pUCP~lacZ210-CEA-I. -CEA-I1 and -CEA-111-M was constructed as follows. The PUCPL-CEAwas digested with BstX I, blunt ended with T4
Two B s t X I (blunt) fragments (530-bp). were inserted into the blunt ended Sac I site of the PUCPL-CIto yield PUCPL-CEA-Iand PUCPL-CEA-11. The BstX I (blunt)-
DNA polymerase and then digested with Xba I.
Xba I fragment (830-bp) was inserted into the Sac I (blunt) and Xba I sites of the PUCPL-CI to yield ~UCPL-CEA-111-M. pGH,650E which
contain the l a c 2 gene and the EcoR I site j u s t before the initiative methionine (our unpublished vector) was digested with Aat 11, blunt ended with T4 DNA polymerase and then digested with EcoR I. The PUCPL-CEA-I was digested with &OR I, blunt ended with T4 DNA polymerase and then digested with B a m H I. The excised EcoR I-Aat I1 (blunt) 640-bp fragment and EcoR I (blunt)-BamH I 430-bp fragment was inserted into the EcoR I and BamH I sites of the PUCPL-CI to yield the pUCP~lacZ2 10-CEA-I.
The pUCPLlacZ210-CEA-I was digested
completely with Xba I and partially with EcoR I and the large fragment was isolated to remove the CEA domain I region. The PUCPL-CEA-I1and PUCPL-CEA-111-Mwere digested with EcoR I and the Xba I and the each excised fragment was ligated to the large fragment to yield the
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pUCP~lacZ210-CEA-11 and -CEA-111-M. The ~UCPLZUCZ~~O-CEA-I, -11
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and -111-M have the open reading frame of the p-galactosidase (residues 1 to 210) followed by artificial linker sequence of EFM and then by CEA domain I (177 amino acids: from Val123 to As11299 ), domain I1 (176 amino acids: from Val301 to Ser476) and domain 111-M (190 amino acids: from Val479 to Ile668). respectively. The pUCP~lacZ210-CEA-I and -CEA-I1 have the additional 16- and 17-residue amino acid sequences at the C-termini (GTRGSSRVDLQACLIN and NGTRGSSRVDLQACLIN, respectively). Each DNA construct was verified by multiple restriction enzymes and partial DNA sequencing. Expression of CEA domains in E. coli. All expression vectors were introduced in E. coli W3110 and the resultant transformants were cultured in SB medium at 32°C. At the late log phase, temperature was shifted to 42OC and the culture was continued for 2 hours to produce the CEA domains under the control of PL promoter, and then the cells
were centrifuged at 5,OOOg for 10 min. washed once with Dulbecco's phosphate-buffered saline free from Ca2+ and Mg2+, and resuspended in the same buffer. Then the cells were disrupted using the French Pressure Cell Press (SLM Instruments, Inc., Urbana, IL) and centrifuged for 10 min at 4,0009, and then the pellets which contained inclusion bodies were frozen at -7OOC. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDSPAGE). Samples were diluted with 0.125 M Tris-HC1 buffer, pH 6.8, containing 4% SDS. 20% glycerol and 10% 2-mercaptoethanol. Electrophoresis was carried out in 4-20% polyacrylamide gels according to the method of Laemmli (30). Proteins in the gels were visualized by staining with Coomassie Blue FU50 or by Western blotting (see below). Western Blotting Analysis. Proteins separated by SDS-PAGE were transferred to hydrophobic Durapore membranes (Millipore, Bedford, MA) as previously described (24,311. The blots were then treated with 10% skim milk, washed with 0.01 M borate-buffered saline (BBS). pH 8.0, containing 0.05% Nonidet P-40, and incubated with a rabbit antiCEA antibody at a concentration of 1 pg/ml for 2 h at room temperature with gentle agitation. After washing, the blots were successively incubated at room temperature with biotinylated goat anti-rabbit IgG (H+L) (0.5 pg/ml) for 1 h, with horseradish peroxidase-avidin D (0.25 pg/ml) for an additional 1 h. and then with 0.01 M Tris-HC1 buffer, pH
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7.4,containing 0.03% 3. 3’-diaminobenzidine and 0.01% H202 for 20 to 30 min. The final color reaction was terminated by washing the filter. Elution of Proteins from Gels. The proteins of interest in the preparative gels were electrically eluted using Model 422 Electroeluter (Bio-Rad Lab., Richmond, CA) with 0.025 M Tris-glycine buffer containing 1% SDS, pH 8.5 (32). The appropriate region on the preparative gel was cut into approximately 1- to 5-mm2 pieces and these were placed in each glass tubes (each 1 x 6 cm longl in which porous polypropylene frits were inserted flush with the base. Elution was done a t 10 mA/glass tube for 5 h. The proteins of interest were retained by the membrane caps with a molecular weight cut off of 3,500, which were connected to the bottom of the glass tube using silicon adaptor. The protein solutions in the membrane caps were carefully aspirated and exhaustively dialyzed against 0.1 M sodium phosphate buffer (NaPB), pH 7.0 containing 0.02% azide. Removal of SDS from Proteins. Removal of SDS from proteins in NaPB was achieved by column chromatography using the ion-retardation resin AG11A8 (Bio-Rad Lab.) (33). About 8.0 g of wet resin was packed into columns (each 1.0 x 10 cm). respectively, and 5 g of swollen Sephadex G-10 was directly layered on the resin columns. The protein solutions were separately passed over these columns at a flow rate of 20 ml/h. and the effluent was collected as one large fraction and concentrated under vacuum using Collodion bag with simultaneous dialysis against BBS. Protein Determination. The protein concentrations were determined by the method of Lowry et al. (34). Solid-phase enzyme immunoassays (SPEIAs). The reactivities of MAbs with the reference antigens or purified recombinant CEA-proteins were estimated by SPEIAs by using the antigens immobilized on 96-well polyvinyl chloride microtiter plates (Dynatech. Alexandria, VA) as described previously (24). Briefly, to equalize the number of moles of antigen, the amount of the purified recombinant proteins immobilized on the 96-well plates were determined from their molecular weights (see below) as follows: 5 ng/50 pl/well for CEA-N: 20 ng for p-GalCEA-I, p-Gal-CEA-11, and p-Gal-CEA-111-M. Fifty pl of each recombinant protein solution in BBS were dried in each well overnight a t 37°C. After blocking with Block Ace (Dainihon Chemical Industries, Ltd.. Osaka, Japan) and washing with 0.05% Nonidet P-40in BBS, varying amounts
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of purified antibodies (up to 10 pg/ml) in 100 pl of 1% BSA in BBS were added and incubated for 1 h a t 37°C. After washing, the plates were successively incubated with biotinylated horse anti-mouse IgG (H+L) antibody (Vector. Burlingame. CAI for 1 h at 37°C. with horseradish peroxidase- avidin D (Vector) for 30 min at room temperature, and then with 150 pl of 0.05 M citrate/O.l M phosphate buffer, pH 5.0. containing 4% o-phenylenediamine and 0.006% H202 for 25 min a t room temperature. The final reaction was terminated by adding 20 pl of 8 N H2W4 and the absorbance at 492 nm of each well was measured.
RESULTS
Production and purification of recombinant proteins. The inclusion bodies containing the respective recombinant proteins were suspended in 3 ml of 0.05M Ms-HCl buffer, pH 8.0, containing 1mM EDTA, 1 mM phenylmethylsulphonyl fluoride and 100 mM NaC1, and treated with lysozyme (260 pg/ml) on ice for 20 min (35). The samples were sonicated (2 x 45 s) on ice and centrifuged at 12,0009 for 15 min at 4°C. The precipitates, which included the fusion products, were resuspended in 1 ml of 0.05M Ws-HCl buffer. Equal volume of 2.0% SDS was added and the samples were resonicated on ice (35).After centrifugation at 14,0009for 30 min. the supernatants were assayed for the presence of fusion proteins by SDS-PAGE. The analytical SDS-PAGE was stained so that locations of specific proteins were visualized by an immunostaining with a specific antibody of electroblotted proteins on a Durapore membrane. The equivalent region on the preparative gel were excised and cut into small pieces. The proteins in the cut gels were electrically eluted using a n electroeluter as described in Materials and Methods (32). Effective removal of SDS from proteins in NaPB was achieved by column chromatography using the ion-retardation resin (33). In Figure 1, SDS-PAGE and Western blotting analyses of each recombinant CEA-protein are demonstrated: they were visualized by staining with Coomassie Blue W 5 0 or by using an affinity-purified rabbit anti-CEA antibody. The apparent molecular weight was 12,800 for CEA-N. 45.000 for p-Gal-CEA-I, 46,000for p-Gal-CEA-11, and 50,000for p-Gal-CEA-III-M (Table I).
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KUROKI ET AL.
FIGURE I SDS-PAGE and Western blotting analyses of the recombinant CEA proteins. Ten pg of each crude extract (Lanes 1. 4. 7. and 10) and two hundred-fifty nanograms each of purified antigen (Lanes 2. 3, 5. 6, 8,9. 11, and 12) were added to each lane. After electrophoresis, the proteins were stained with Coomassie blue R250 (Lanes 1, 2. 4, 5, 7, 8. 10 and 1 1). or transferred to hydrophobic Durapore filter, and immunostained with an affinity-purified rabbit anti-CEA antibody (Lanes 3, 6, 9. and 12) as described in "Material and Methods". Vertical scales, mol. wt markers IX 10-3).
Immunoreactivity of the Purified Recombinant Proteins. Immunoreactivities of the purified recombinant proteins were tested by SPEIAs against 21 different MAbs which all had been shown to recognize the protein epitopes of the CEA molecule, respectively, and categorized into six groups A-F according to their reactivity with the CEA-related antigens (18) and different domains of the CEA or NCA molecule expressed in CHO cells (Table 11) (24). Those are: 2 Group A MAbs recognizing epitopes common to CEA and NCA. and present on the domain N of the CEA and NCA molecules: 2 Group B MAbs against epitopes also shared between CEA and NCA. but present on the domain I of the CEA and NCA molecules: 4 Group C, 5 Group D and 6 Group E MAbs recognizing epitopes absent from NCA but present on CEA and normal fecal antigens (WAS) (27.37).and present on the domain N, I.
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TABLE I Structures and molecular weights of recombinant proteins expressed in E. colt Recombinant protein
Organization of chimeric proteina
Calculated mol. wt
Estimated mol. wt
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~
CEA-N
CEA-N 11-1061
p-Gal-CEA-I (A1-B 1)
+ (6)
~
12.531
12,800
P-Gal + CEA-I (123-2991 + (16)
45.736
45,000
P-Gal-CEA-I1 (A2-B2)
@-Gal+ CEA-I1 (301-4761 + (17)
45,676
46.000
p-Gal-CEA-111-Mb (A3-B3)
P-Gd
44.661
50.000
+ 111-M [479-6681
acid positions for CEA are in [ I. Numbers in ( ) are amino acid residues attached for the convenience of construction of expression vectors. p-Gal is amino acids 1 to 210 of P-galactosidase and a hipeptide linker as described in Materials and Methods. b The product retains M-domain.
a Amino
and I1 of the CEA molecule, respectlvely; and 2 Group F MAbs directed against epitopes which had been known to be present on CEA but not on NCA or NFAs, and previously been described as "CEA distinctive" (181, and have recently been found to be located on the domain 111 of the CEA molecule (24). The titration curves were carried out for all 21 MAbs versus the 4 purified recombinant CEA-proteins, CEA-N. p-Gal-CEA-I, P-Gal-CEA-11. and p-Gal-CEA-111-M: representative curves are shown in Figure 2. An affinity-purified BALB/c polyclonal anti-CEA antibody reacted well with all 4 recombinant proteins (Figures 2A-D). F34-187 of Group A and F4-82 of Group C reacted with CEA-N [Figure 2A) but did not with any of other 3 proteins (Figures 2B. 2C and 2D). F36-54 of Group B and F33-13 of Group D reacted only with p-Gal-CEA-I. but the reactivity of the latter was one third of that of the former (Figure 2B). A Group D MAb, F36-14, reacted with p-Gal-CEA-I (Figure 2B) and also 0-Gal-CEA-I1
KUROKI ET AL.
2 50
L . "
I A.CEA-N
-
'
N
2.0
B. p-Gal-CEA-I
1.5
0)
t UJ
y
1.0
d m
a
8 0.5 m d
.u "
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0.3
o.d
40
176 8.0 200 1,000 ANTIBODY ADDED (ng)
0.3
1.6
8.0
40
200
0
8%
40
200 1,000
ANTIBODY ADDED (ng)
1,000 ANTIBODY ADDED (ng)
ANTIBODY ADDED (ng)
I
1.6
Ab used: X, Polyclonal A b 0, F34-187; 0,F36-54; A ,F4-82;
.
A F33-13: U. F36-14: M.F3-30:
0. F19-109;
,F36-96
I
FIGURE 2 Reactivities of 8 different MAbs with recombinant CEA proteins in SPEIAs. According to the mol. wts of the purified recombinant proteins, five to twenty nanograms of each antigen per 50 p1 were dried onto wells of 96-well plates and assayed for each MAb as described in "Materials and Methods".
(Figure 2C). but did not with either CEA-N or p-Gal-CEA-111-M (Figures 2A and 2D). F3-30 of Group E reacted well with p-Gal-CEA-I1 (Figure 2C) and fairly with p-Gal-CEA-111-M (Figure 2D). but neither with CEA-N (Figure 2A1 nor with p-Gal-CEA-I (Figure 2B). Thus these 2 MAbs (F3614 and F3-30) showed positive reactions with two different domains. A Group E MAb, F19-109. showed a weak reaction with p-Gal-CEA-I1 (Figure 2C). but did not with any of other 3 proteins (Figures 2A, 2B and 2D). F36-96 of Group F weakly reacted only with p-Gal-CEA-111-M (Figure 2D).
RECOMBINANT CARCINOEMBRYONIC ANTIGEN PROTEINS
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TABLE I1 Reactivities of recombinant CEA proteins expressed in E. coli with 21 different anti-CEA MAbs determined by SPElAsa Antibody
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Group
Epitope localizationb
Clone
F34- 187[5]d N F36-6 1 N Bc F36-54 I F14-100 I C F4-82[5] N F33-49 N F82-68 N F82-6 1 N D F33- 13 I F36-68 I F33-20 I F36- 14 I F82-2 1 I E F3-30 I1 F84-46[1] I1 F4-95 I1 F6-2 I1 F19- 109 I1 F36-65 I1 F F36-96 I11 F33- 104[11 I11 Mouse polyclonal anti-CEA ~
~
p-GalCEA-I
CEA-N
p-Gal- p-GalCEA-I1 CEA-111-M
2+e 2+
AC
~
CEA protein expressed in E. coli
2+ -f
2+ 2+ 2+ 2+ +f
2+ 2+ 2+
2+
-f
2+ 2+ 2+ 2+
+
+f
+f *f +f
2+
2+ ~
~
~
2+
2+
~~
The reactivity of recombinant CEA proteins was determined by SPEIAs as described in "Materials and Methods". b Determined by reactivities of the recombinant CEA or NCA proteins expressed in CHO cells (24). c Only Groups A and B anti-CEA MAbs were cross-reactive with NCA. d The groups in the Gold classification system determined in the international workshop (22) were also indicated for four MAbs with the bold numbers in bold face brackets. e The degree of the absorbance at 492 nm obtained at the highest concentration of MAb used (1 pg/lOO pl/well) was classified into 5 groups. The absorbance obtained by the use of MOPC 21 (IgG1) or MOPC104E (IgM) instead of MAbs was subtracted from the absorbance obtained with MAbs. The code is a s follows: 2+, 1.0 SA492 < 2.0: +. 0.5I&m
INVESTIGATIONS, 21(3), 241-257 (1992)
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IMMUNOREACI'MTY OF RECOMBINANT CARCINOEMBRYONIC ANTIGEN PROTEINS EXPRESSED IN ESCHEZUCHIA COLJ Masahide Kuroki, Masaaki Murakami. Mamie Wakisaka. Shoichi Ikeda, Shinu, Oikawal , Takehiro Oshimal. Hiroshi Nakazatol , Goro Kosaki2 and Yuji Matsuoka First Department of Biochemistry, School of Medicine, Fukuoka University, 7-45- I Nanakuma, Jonan-ku. Fukuoka 814-01, Japan 1Suntory Institute for Biomedical Research. 1- 1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 6 18, Japan. Tokyo Metropolitan Komagome Hospital, Tokyo 170, Japan
Immunoreactivities of recombinant carcinoembryonic antigen (CEA) proteins expressed in Escherichia coli (E. coli) were analyzed in relation to the CEA domain structure [domains N, I (Al-Bl). I1 (A2-B2), I11 (A3-B3) and MI. We reconstructed in a prokaryotic expression vector, PUCPL-cI. the cDNAs for CEA-N, CEA-I. CEA-11. and CEA-111-M. The latter three were expressed as fusion products with bacterial p-galactosidase. The recombinant proteins were s o l u b i k d by sonication in 1% sodium dodecyl sulfate (SDS) and purified by preparative SDS-polyacrylamide gel electrophoresis followed by electroelution. Their molecular weights judged from Western blotting coincided with those calculated from their cDNA sequences, respectively. By solidphase enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested with 21 distinct anti-CEA monoclonal antibodies (MAbs) which had been found to recognize the peptide epitopes of the CEA molecule and to be reactive with the recombinant CEA proteins expressed in Chinese hamster ovary (CHO) cells. Fourteen of the 21 MAbs reacted with the recombinant CEA proteins expressed in E. colt and confirmed the localization of the epitopes identified by using the recombinant CEA proteins expressed in CHO cells. The reactivities of 5 MAbs with the recombinant proteins expressed in E. coli were remarkably low when compared with those of the proteins expressed in CHO cells but also confirmed the localization of the 241
Copyright 0 1992 by Marcel Dekker, Inc.
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epitopes identified with the recombinant CEA proteins expressed in CHO cells. The remaining 2 M A b s did not react with any recombinant protein expressed in E. coli. These results indicate that the fusion CEA-proteins expressed in E. coli are useful in the localization of the epitopes on the polypeptide chains when they reacted with the MAbs tested. However, one third of the epitopes of CEA peptides may be profoundly affected by the presence of disulfide bonds and/or sugar chains which do not seem to be formed well in E. coli.
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INTRODUCTION
Carcinoembryonic antigen (CEA) is a glycoprotein with a molecular weight of 180,000 and is one of the most useful human tumor markers for the post surgical monitoring of patients with various malignancies (1). In addition there is increasing interest in the in uiuo use of anti-CEA MAbs in tumor imaging (2) or therapy (3). The primary structure of CEA peptide. as determined by chemical sequence analysis (4) and successful cDNA cloning (5). has revealed that it is a member of the CEA gene family within immunoglobulin supergene family (4. 6). I t has been known that the CEA gene family consists of at least 20 different genes (7).The CEA structure is composed of the following five domains (5): a n N-terminal domain (N) of 108 residues: three very homologous repetitive domains I, 11. and 111 of 178 residues, each of which was subdivided into A and B subdomains and designated A1-B1, A2-B2, and A3-B3, respectively, by the newly proposed nomenclature (8);and a hydrophobic C-terminal domain (abbreviated as M) of 26 residues which are post-translationally replaced with a glycosylphosphatidylinositol (GPI) moiety for membrane anchoring (9.10). Furthermore, several recent works have clarified that CEA has cell adhesion activity (11.12) or bacterial binding activity (13.14). These findings suggest that it might play some important role in cell-cell interactions. Since more than 20 structurally related proteins, known to be CEArelated antigens, have been found in various human tissues or body fluids as members of the CEA gene family (7).elucidation of the specificity of MAbs used to detect CEA is very important. The specificity of anti-CEA MAbs has been evaluated by several approaches including the analysis of cross-reactivity with the CEA-related antigens (15-18). immunohistochemical reactivity with a variety of normal or malignant tissues (19-211, and competitive binding activity among MAbs (22). Recently.
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243
recombinant CEA proteins including fusion proteins have been produced using eukaryotic (23.24) or prokaryotic (25) expression vectors and were utilized for analyzing the specificity of anti-CEA MAbs (24.25). In recent works, we prepared several recombinant CEA- or NCA-proteins with different domain structures by expressing the corresponding genes in Chinese hamster ovary (CHO) cells, and mapped the epitopes recognized by 21 different anti-CEA (24) or 8 different anti-NCA MAbs (26) in terms of domain structure. In the present study, we expressed the fusion proteins composed of bacterial p-galactosidase and each of the 4 principal domains of CEA in E. coli, and demonstrated the utility of these proteins in mapping the epitopes of the CEA molecule. The results were compared to our previous mapping obtained with recombinant CEA-proteins expressed in CHO cells.
MATERIALS AND METHODS
Antibodies. Polyclonal anti-CEA antisera were prepared in rabbits or BALB/c mice as described previously (27.28). and specifically purified with CEA-Sepharose 4B. respectively (28). The affinity-purified BALB/c polyclonal anti-CEA antibody was used as positive control. Twenty-one different anti-CEA MAbs used in this study were prepared and purified as described elsewhere (16.18). Purified myeloma proteins MOPC 21 (IgG1) and MOPC 104E (IgM) were used as negative controls (Organon Technika, West Chester, PA). Reference CEA and NCA Preparations. Reference CEA was purified from a metastatic liver tumor from a colonic adenocarcinoma by the procedure described earlier (29). Reference NCA was purified from pooled normal human lungs by the procedures described previously (28). Construction of prokaryotic expression vectors. The expression vector used in this study was the PUCPL-CI which contains a PL promoter, cIts857, Shine-Dalgarno sequence, initiative methionine. multi-cloning sites, stop codons in any frame, and trp terminator (our unpublished vector). Restriction enzymes were purchased from Takara Shuzo (Tokyo, Japan) except for BsfX I (New England Nuclear, Boston, MA).
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PUCPL-CEA-Nwas constructed as follows. The pCEA55-2 cloned in Okayama-Berg vector (5) was digested with Hind I11 and Dra I. then excised fragment was inserted into the Hind I11 and Hfnc I1 sites of the M 13mp18 to yield a single-strand DNA template (M 13mp 18CEA55-2) for site-directed mutagenesis. The M 13mp18CEA668-8 which h a s the EcoR I site and the initiative methionine j u s t before the N-terminal amino acid, lysine, of the CEA. was produced with 33-mer oligonucleotide, 5-TGGAACCCGCCCGAA'ITCATGAAGCTCACTAlT-3'using the oligonucleotide-directed in uitro mutagenesis system (Amersham. Buckinghamshire. England) and then digested with the EcoR I and Sma I, then the excised fragment was inserted into the EcoR I and Sma I sites of the PUCPL-CIto yield PUCPL-CEA. The PUCPL-CEAwas digested with Acc I, blunt ended with T4 DNA polymerase and the large fragment was self-ligated to yield the ~UCPL-CEA-Nwhich h a s the open reading
frame of the initiative methionine followed by CEA-N-domain (106 amino acids, from Lysl to VallO6) with a n additional artificial six-amino acid sequence. STCRHA a t the C-terminus. pUCP~lacZ210-CEA-I. -CEA-I1 and -CEA-111-M was constructed as follows. The PUCPL-CEAwas digested with BstX I, blunt ended with T4
Two B s t X I (blunt) fragments (530-bp). were inserted into the blunt ended Sac I site of the PUCPL-CIto yield PUCPL-CEA-Iand PUCPL-CEA-11. The BstX I (blunt)-
DNA polymerase and then digested with Xba I.
Xba I fragment (830-bp) was inserted into the Sac I (blunt) and Xba I sites of the PUCPL-CI to yield ~UCPL-CEA-111-M. pGH,650E which
contain the l a c 2 gene and the EcoR I site j u s t before the initiative methionine (our unpublished vector) was digested with Aat 11, blunt ended with T4 DNA polymerase and then digested with EcoR I. The PUCPL-CEA-I was digested with &OR I, blunt ended with T4 DNA polymerase and then digested with B a m H I. The excised EcoR I-Aat I1 (blunt) 640-bp fragment and EcoR I (blunt)-BamH I 430-bp fragment was inserted into the EcoR I and BamH I sites of the PUCPL-CI to yield the pUCP~lacZ2 10-CEA-I.
The pUCPLlacZ210-CEA-I was digested
completely with Xba I and partially with EcoR I and the large fragment was isolated to remove the CEA domain I region. The PUCPL-CEA-I1and PUCPL-CEA-111-Mwere digested with EcoR I and the Xba I and the each excised fragment was ligated to the large fragment to yield the
RECOMBINANT CARCINOEMBRYONIC ANTIGEN PROTEINS
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pUCP~lacZ210-CEA-11 and -CEA-111-M. The ~UCPLZUCZ~~O-CEA-I, -11
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and -111-M have the open reading frame of the p-galactosidase (residues 1 to 210) followed by artificial linker sequence of EFM and then by CEA domain I (177 amino acids: from Val123 to As11299 ), domain I1 (176 amino acids: from Val301 to Ser476) and domain 111-M (190 amino acids: from Val479 to Ile668). respectively. The pUCP~lacZ210-CEA-I and -CEA-I1 have the additional 16- and 17-residue amino acid sequences at the C-termini (GTRGSSRVDLQACLIN and NGTRGSSRVDLQACLIN, respectively). Each DNA construct was verified by multiple restriction enzymes and partial DNA sequencing. Expression of CEA domains in E. coli. All expression vectors were introduced in E. coli W3110 and the resultant transformants were cultured in SB medium at 32°C. At the late log phase, temperature was shifted to 42OC and the culture was continued for 2 hours to produce the CEA domains under the control of PL promoter, and then the cells
were centrifuged at 5,OOOg for 10 min. washed once with Dulbecco's phosphate-buffered saline free from Ca2+ and Mg2+, and resuspended in the same buffer. Then the cells were disrupted using the French Pressure Cell Press (SLM Instruments, Inc., Urbana, IL) and centrifuged for 10 min at 4,0009, and then the pellets which contained inclusion bodies were frozen at -7OOC. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDSPAGE). Samples were diluted with 0.125 M Tris-HC1 buffer, pH 6.8, containing 4% SDS. 20% glycerol and 10% 2-mercaptoethanol. Electrophoresis was carried out in 4-20% polyacrylamide gels according to the method of Laemmli (30). Proteins in the gels were visualized by staining with Coomassie Blue FU50 or by Western blotting (see below). Western Blotting Analysis. Proteins separated by SDS-PAGE were transferred to hydrophobic Durapore membranes (Millipore, Bedford, MA) as previously described (24,311. The blots were then treated with 10% skim milk, washed with 0.01 M borate-buffered saline (BBS). pH 8.0, containing 0.05% Nonidet P-40, and incubated with a rabbit antiCEA antibody at a concentration of 1 pg/ml for 2 h at room temperature with gentle agitation. After washing, the blots were successively incubated at room temperature with biotinylated goat anti-rabbit IgG (H+L) (0.5 pg/ml) for 1 h, with horseradish peroxidase-avidin D (0.25 pg/ml) for an additional 1 h. and then with 0.01 M Tris-HC1 buffer, pH
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7.4,containing 0.03% 3. 3’-diaminobenzidine and 0.01% H202 for 20 to 30 min. The final color reaction was terminated by washing the filter. Elution of Proteins from Gels. The proteins of interest in the preparative gels were electrically eluted using Model 422 Electroeluter (Bio-Rad Lab., Richmond, CA) with 0.025 M Tris-glycine buffer containing 1% SDS, pH 8.5 (32). The appropriate region on the preparative gel was cut into approximately 1- to 5-mm2 pieces and these were placed in each glass tubes (each 1 x 6 cm longl in which porous polypropylene frits were inserted flush with the base. Elution was done a t 10 mA/glass tube for 5 h. The proteins of interest were retained by the membrane caps with a molecular weight cut off of 3,500, which were connected to the bottom of the glass tube using silicon adaptor. The protein solutions in the membrane caps were carefully aspirated and exhaustively dialyzed against 0.1 M sodium phosphate buffer (NaPB), pH 7.0 containing 0.02% azide. Removal of SDS from Proteins. Removal of SDS from proteins in NaPB was achieved by column chromatography using the ion-retardation resin AG11A8 (Bio-Rad Lab.) (33). About 8.0 g of wet resin was packed into columns (each 1.0 x 10 cm). respectively, and 5 g of swollen Sephadex G-10 was directly layered on the resin columns. The protein solutions were separately passed over these columns at a flow rate of 20 ml/h. and the effluent was collected as one large fraction and concentrated under vacuum using Collodion bag with simultaneous dialysis against BBS. Protein Determination. The protein concentrations were determined by the method of Lowry et al. (34). Solid-phase enzyme immunoassays (SPEIAs). The reactivities of MAbs with the reference antigens or purified recombinant CEA-proteins were estimated by SPEIAs by using the antigens immobilized on 96-well polyvinyl chloride microtiter plates (Dynatech. Alexandria, VA) as described previously (24). Briefly, to equalize the number of moles of antigen, the amount of the purified recombinant proteins immobilized on the 96-well plates were determined from their molecular weights (see below) as follows: 5 ng/50 pl/well for CEA-N: 20 ng for p-GalCEA-I, p-Gal-CEA-11, and p-Gal-CEA-111-M. Fifty pl of each recombinant protein solution in BBS were dried in each well overnight a t 37°C. After blocking with Block Ace (Dainihon Chemical Industries, Ltd.. Osaka, Japan) and washing with 0.05% Nonidet P-40in BBS, varying amounts
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of purified antibodies (up to 10 pg/ml) in 100 pl of 1% BSA in BBS were added and incubated for 1 h a t 37°C. After washing, the plates were successively incubated with biotinylated horse anti-mouse IgG (H+L) antibody (Vector. Burlingame. CAI for 1 h at 37°C. with horseradish peroxidase- avidin D (Vector) for 30 min at room temperature, and then with 150 pl of 0.05 M citrate/O.l M phosphate buffer, pH 5.0. containing 4% o-phenylenediamine and 0.006% H202 for 25 min a t room temperature. The final reaction was terminated by adding 20 pl of 8 N H2W4 and the absorbance at 492 nm of each well was measured.
RESULTS
Production and purification of recombinant proteins. The inclusion bodies containing the respective recombinant proteins were suspended in 3 ml of 0.05M Ms-HCl buffer, pH 8.0, containing 1mM EDTA, 1 mM phenylmethylsulphonyl fluoride and 100 mM NaC1, and treated with lysozyme (260 pg/ml) on ice for 20 min (35). The samples were sonicated (2 x 45 s) on ice and centrifuged at 12,0009 for 15 min at 4°C. The precipitates, which included the fusion products, were resuspended in 1 ml of 0.05M Ws-HCl buffer. Equal volume of 2.0% SDS was added and the samples were resonicated on ice (35).After centrifugation at 14,0009for 30 min. the supernatants were assayed for the presence of fusion proteins by SDS-PAGE. The analytical SDS-PAGE was stained so that locations of specific proteins were visualized by an immunostaining with a specific antibody of electroblotted proteins on a Durapore membrane. The equivalent region on the preparative gel were excised and cut into small pieces. The proteins in the cut gels were electrically eluted using a n electroeluter as described in Materials and Methods (32). Effective removal of SDS from proteins in NaPB was achieved by column chromatography using the ion-retardation resin (33). In Figure 1, SDS-PAGE and Western blotting analyses of each recombinant CEA-protein are demonstrated: they were visualized by staining with Coomassie Blue W 5 0 or by using an affinity-purified rabbit anti-CEA antibody. The apparent molecular weight was 12,800 for CEA-N. 45.000 for p-Gal-CEA-I, 46,000for p-Gal-CEA-11, and 50,000for p-Gal-CEA-III-M (Table I).
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FIGURE I SDS-PAGE and Western blotting analyses of the recombinant CEA proteins. Ten pg of each crude extract (Lanes 1. 4. 7. and 10) and two hundred-fifty nanograms each of purified antigen (Lanes 2. 3, 5. 6, 8,9. 11, and 12) were added to each lane. After electrophoresis, the proteins were stained with Coomassie blue R250 (Lanes 1, 2. 4, 5, 7, 8. 10 and 1 1). or transferred to hydrophobic Durapore filter, and immunostained with an affinity-purified rabbit anti-CEA antibody (Lanes 3, 6, 9. and 12) as described in "Material and Methods". Vertical scales, mol. wt markers IX 10-3).
Immunoreactivity of the Purified Recombinant Proteins. Immunoreactivities of the purified recombinant proteins were tested by SPEIAs against 21 different MAbs which all had been shown to recognize the protein epitopes of the CEA molecule, respectively, and categorized into six groups A-F according to their reactivity with the CEA-related antigens (18) and different domains of the CEA or NCA molecule expressed in CHO cells (Table 11) (24). Those are: 2 Group A MAbs recognizing epitopes common to CEA and NCA. and present on the domain N of the CEA and NCA molecules: 2 Group B MAbs against epitopes also shared between CEA and NCA. but present on the domain I of the CEA and NCA molecules: 4 Group C, 5 Group D and 6 Group E MAbs recognizing epitopes absent from NCA but present on CEA and normal fecal antigens (WAS) (27.37).and present on the domain N, I.
RECOMBINANT CARCINOEHBRYONIC ANTIGEN PROTEINS
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TABLE I Structures and molecular weights of recombinant proteins expressed in E. colt Recombinant protein
Organization of chimeric proteina
Calculated mol. wt
Estimated mol. wt
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~
CEA-N
CEA-N 11-1061
p-Gal-CEA-I (A1-B 1)
+ (6)
~
12.531
12,800
P-Gal + CEA-I (123-2991 + (16)
45.736
45,000
P-Gal-CEA-I1 (A2-B2)
@-Gal+ CEA-I1 (301-4761 + (17)
45,676
46.000
p-Gal-CEA-111-Mb (A3-B3)
P-Gd
44.661
50.000
+ 111-M [479-6681
acid positions for CEA are in [ I. Numbers in ( ) are amino acid residues attached for the convenience of construction of expression vectors. p-Gal is amino acids 1 to 210 of P-galactosidase and a hipeptide linker as described in Materials and Methods. b The product retains M-domain.
a Amino
and I1 of the CEA molecule, respectlvely; and 2 Group F MAbs directed against epitopes which had been known to be present on CEA but not on NCA or NFAs, and previously been described as "CEA distinctive" (181, and have recently been found to be located on the domain 111 of the CEA molecule (24). The titration curves were carried out for all 21 MAbs versus the 4 purified recombinant CEA-proteins, CEA-N. p-Gal-CEA-I, P-Gal-CEA-11. and p-Gal-CEA-111-M: representative curves are shown in Figure 2. An affinity-purified BALB/c polyclonal anti-CEA antibody reacted well with all 4 recombinant proteins (Figures 2A-D). F34-187 of Group A and F4-82 of Group C reacted with CEA-N [Figure 2A) but did not with any of other 3 proteins (Figures 2B. 2C and 2D). F36-54 of Group B and F33-13 of Group D reacted only with p-Gal-CEA-I. but the reactivity of the latter was one third of that of the former (Figure 2B). A Group D MAb, F36-14, reacted with p-Gal-CEA-I (Figure 2B) and also 0-Gal-CEA-I1
KUROKI ET AL.
2 50
L . "
I A.CEA-N
-
'
N
2.0
B. p-Gal-CEA-I
1.5
0)
t UJ
y
1.0
d m
a
8 0.5 m d
.u "
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0.3
o.d
40
176 8.0 200 1,000 ANTIBODY ADDED (ng)
0.3
1.6
8.0
40
200
0
8%
40
200 1,000
ANTIBODY ADDED (ng)
1,000 ANTIBODY ADDED (ng)
ANTIBODY ADDED (ng)
I
1.6
Ab used: X, Polyclonal A b 0, F34-187; 0,F36-54; A ,F4-82;
.
A F33-13: U. F36-14: M.F3-30:
0. F19-109;
,F36-96
I
FIGURE 2 Reactivities of 8 different MAbs with recombinant CEA proteins in SPEIAs. According to the mol. wts of the purified recombinant proteins, five to twenty nanograms of each antigen per 50 p1 were dried onto wells of 96-well plates and assayed for each MAb as described in "Materials and Methods".
(Figure 2C). but did not with either CEA-N or p-Gal-CEA-111-M (Figures 2A and 2D). F3-30 of Group E reacted well with p-Gal-CEA-I1 (Figure 2C) and fairly with p-Gal-CEA-111-M (Figure 2D). but neither with CEA-N (Figure 2A1 nor with p-Gal-CEA-I (Figure 2B). Thus these 2 MAbs (F3614 and F3-30) showed positive reactions with two different domains. A Group E MAb, F19-109. showed a weak reaction with p-Gal-CEA-I1 (Figure 2C). but did not with any of other 3 proteins (Figures 2A, 2B and 2D). F36-96 of Group F weakly reacted only with p-Gal-CEA-111-M (Figure 2D).
RECOMBINANT CARCINOEMBRYONIC ANTIGEN PROTEINS
251
TABLE I1 Reactivities of recombinant CEA proteins expressed in E. coli with 21 different anti-CEA MAbs determined by SPElAsa Antibody
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Group
Epitope localizationb
Clone
F34- 187[5]d N F36-6 1 N Bc F36-54 I F14-100 I C F4-82[5] N F33-49 N F82-68 N F82-6 1 N D F33- 13 I F36-68 I F33-20 I F36- 14 I F82-2 1 I E F3-30 I1 F84-46[1] I1 F4-95 I1 F6-2 I1 F19- 109 I1 F36-65 I1 F F36-96 I11 F33- 104[11 I11 Mouse polyclonal anti-CEA ~
~
p-GalCEA-I
CEA-N
p-Gal- p-GalCEA-I1 CEA-111-M
2+e 2+
AC
~
CEA protein expressed in E. coli
2+ -f
2+ 2+ 2+ 2+ +f
2+ 2+ 2+
2+
-f
2+ 2+ 2+ 2+
+
+f
+f *f +f
2+
2+ ~
~
~
2+
2+
~~
The reactivity of recombinant CEA proteins was determined by SPEIAs as described in "Materials and Methods". b Determined by reactivities of the recombinant CEA or NCA proteins expressed in CHO cells (24). c Only Groups A and B anti-CEA MAbs were cross-reactive with NCA. d The groups in the Gold classification system determined in the international workshop (22) were also indicated for four MAbs with the bold numbers in bold face brackets. e The degree of the absorbance at 492 nm obtained at the highest concentration of MAb used (1 pg/lOO pl/well) was classified into 5 groups. The absorbance obtained by the use of MOPC 21 (IgG1) or MOPC104E (IgM) instead of MAbs was subtracted from the absorbance obtained with MAbs. The code is a s follows: 2+, 1.0 SA492 < 2.0: +. 0.5I&m
