- Email: [email protected]
Sequences of the cDNAs encoding the heavy- and light-chain Fab region of an antibody to the phenylurea herbicide diuron
Gene, 165 (1995) 323-324 ©1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50
323
GENE 09246
Sequences of the cDNAs encoding the heavy- and light-chain Fab region of an antibody to the phenylurea herbicide diuron * (Recombinant DNA; complementarity determining region; antibody structure; phage display; mouse hybridoma)
C h r i s t o p h e r W. BelP, K a r e n - B e t h G. S c h o l t h o f
b,**, G u i s h e n g
Z h a n g T M a n d A l e x a n d e r E. K a r u a
"Hybridoma Facility, College of Natural Resources, University of California-Berkeley, Albany, CA 94706, USA; and bDepartment of Plant Biology, University of California-Berkelek; Berkeley, CA 94720, USA Received by C.M. Kane: 22 May 1995; Revised/Accepted: 30 June/5 July 1995; Received at publishers: 3 August 1995
SUMMARY
The cDNAs from a hybridoma (mAb 481.1) specific for diuron, a widely used phenylurea herbicide, were cloned into the phage display vector pComb8. Antigen-binding clones were selected by panning on diuron-hapten-BSA conjugates. The nucleotide and deduced amino-acid sequences encoding the Fab regions of the light (~:) and heavy (5') chains were determined. The light chain was from mouse ~cchain subgroup III and the heavy chain was a member of the mouse H chain subgroup III(d).
Diuron (3-(3,4-dichlorophenyl)-l,l-dimethylurea) is a widely used phenylurea herbicide. We have developed a series of mAb and EIAs that detect parts per billion (ng/ml) of diuron (Karu et al., 1994). We chose to clone the Ab-encoding genes from one of these hybridomas as the first step toward engineering new binding specificities for the phenylureas. We amplified the Fab genes from the cDNA ofmAb 481.1 (an IgG1K), and cloned them into Correspondence to: Dr. A.E. Karu, Hybridoma Facility, College of Natural Resources, University of California-Berkeley, 1050 San Pablo Ave, Albany, CA 94706, USA. Tel. (1-510) 643-7746; Fax (1-510) 642-0875; e-mail: [email protected] **Present addresses: (K-B.G.S.) Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA. Tel. (1-409) 845-8265; (G.Z.) Department of Transplantation Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. Tel. (1-412) 648-2080. * On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note. Abbreviations: aa, amino acid(s); Ab, antibody(ies); bp, base pair(s); BSA, bovine serum albumin; cDNA, DNA complementary to RNA; CDR, complementarity-determining region; C, constant region; EIA, enzyme immunoassay; Fab, antibody-binding fraction(s); FR, framework region; H, heavy; Ig, immunoglobulin; L, light; kb, kilobase(s) or 1000 bp; mAb, monoclonal Ab; nt, nucleotide(s); ORF, open reading frame.
SSDI 0378-1119(95~00531-5
the phage display vector pComb8 (Barbas et al., 1991). Antigen-binding phages were selected by rounds of panning with diuron hapten-BSA conjugates, and positive clones were subcloned into pComb3 (Barbas et al., 1991). Soluble recombinant Fab from these clones competitively bound diuron in EIAs with the same sensitivity and specificity as mAb 481.1 (K-B.G.S., G.Z. and A.E.K., data not shown). Sequences from one of the diuron-specific Fab 481.1 clones (112) are shown in Fig. 1. Each sequence encoded a single ORF with homology to known Ab aa sequences (Kabat et al., 1991). The L chain ORF extends from FR-1 (a SacI site) to the last residue (CysTM) of the CL region, and it is terminated by two in-frame stop codons (encoded within the primers). The H chain ORF begins at aa 6 (an XhoI site) in FR-1, the first five FR residues (AQVKL) being coded by the vector (Barbas et al., 1991 ), and continues to Cysnz35 at the end of Cm in the hinge region. From comparisons with the immunological database of Kabat et al. (1991), the H chain is a member of the mouse H chain subgroup III(d) and the L chain is a member of mouse ~ chain subgroup III. The hypervariable regions or CDRs, which form the antigen-binding loops of the antibody, were also identified (Fig. 1).
324
Fab 481.1 Lc (1<) i
FRI
C~R-LI
,
~R-2
GA$~T~;GTGATGACACAGTCTCCAGCT~GCTGTGTCTCTA~AGAGTGTCACCATCTCCTGCAGAGCCAGTGAAAGTGTTGAATATTATGGCACTAGTTTAATGCAGTC~GTACCAACAGA~%ACCAGGACAGCCACC~CTC
E
L
V
M
T
Q
I
S
P
A
S
L
CDR-L2
A
V
S
L
G
Q
S
V
T
I
S
C
R
A
I
C TCATCTATGGTGCATCCAACGTAGAATCTG la I Y G A S N V E S
S
E
S
V
E
Y
Y
27
a
b
c
d
e
G
T
S
L
M f 28 29 32 33
Q
W
Y
Q
Q
FR-3
K
P
G
150
Q
I
P
P
K
L
46
CDR-L3
G G G T C C C T G C C A G G T T T A G T G GCAGTGC4~ T C T G G G A C A G A C T T C A G C C T C A A C A T C C A T C C T G T G G A G G A G G A T G A T A T T G C A A T A T A T T T C T G T C A G ~GTAGGAAGGTTCCAG G V P A R F S G S G S G T D F S L N I H P V E E D D I A I Y F C Q Q S R K V P
CT A
300 96
C T C A G T C G T G T C47TT C T T G A A C A A C T T C T A C C C C A 2 ~ G A C A T C A A T G T C C F L N N F Y P K D I N V I 4 6
450
AAGTGGAAGATTGATGGCAG TGAACGACAAAAT GGCG TC CTGAACAG TTGGACTGATCAGGAC AGCAAAGACAGCAC CTACAG CA TGAGCAGCACC CT CACGTTGACCAAGGACGAGTATGAACGACATAACAGC TATAC CTGTGAGG CC K W K I D G S E R Q N G V L N S W T D Q D S K D S T Y S M S S T L T L T K D E Y E R H N S Y T C E A
600 196
FR4 I ACGTTCGGATCGGGGAC CAAGCTGGA.%ATAAAACGGGC TGATGCTGCAC CAAC TG TA TCCAT C TTC CCAC CATC CAGTGAGCAGTTAACATCTGGAGGTGC T F G S G T K L E I K R A D A A P T V S I F P P S S E Q L T S G G A S V V
CL
AC TCACAAGACATCAACTTCAC CCATCGTCAAGAGC TTCAACAGGAATGAGTGTTAATT CTAGA T H K T S T S P I V K S F N R N E C *
664 214
Fab 481.1 Hc (7) J~R1 . " CDR-~I "~ FR-2 I C TCGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTTGCATACATTAGTAGTGGC L
E
S
S
S
T
G
G
G
L
V
K
P
G
G
Y
A
D
T
V
K
G
CDR-H2 I
Y
S
L
K
L
S
C
A
A
S
G
F
T
F
S
D
R
F
T
I
S
R
D
N
A
K
N
T
L
F
L
I
Y
G
M
H
W
V
R
Q
A
P
E
K
G
L
E
W
V
M 82
T a
S b
L R c 83
S
E
D
T
A
M
Y
Y
C
A
R
FR-3 Q
A
"/
I
$ 52
S a
I
150 Ca
III W
mmm CDR-H3 I FR-4 I G G T C A T T A T T A T G T T A T G G A C T A C T C - G G G T C A A G G A - % CC T C a ~ G T C A C C G T C T C C T C A G C C A A A A C G A C A C C C C C A T C T G T C T A T C C A C T G G C C C CTC~?~ATC T G C T G C C C A A A C T A A C T C C A T G G T G A C C C T G G G A T G C C T G G ~ C A ~ G G G C G H Y Y V M D Y W G Q G T S V T V S S A K T T P P S V Y P L A P G S A A Q T N S M V T L G C L a
b
c
d
e
53
i00
450 146
k 101
CH-I TAT TTCCCTGAGCCAGTGACAGTGACcTGGAACTCTGGATCCcTGTCCAGcGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACcTCTACACTCTGAGCAGCTCAGTGACTGTCCCCT~CAGCACCTC,GCCCAGCcAGACCGTCACC Y F P E P V T V T W N S G S L S S G v H T F P A V L Q S D L ¥ T L S S S V T %" P S S T W P S Q T it
600 207
I TGCAACGTTGCCCAC CCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGC C N V A H P A S S T K V D K K I V
CCAGGGATTGTACTAGC P R D C T S
669
237
Fig. 1. The nt sequence of diuron-specific Fab 481.1 (clone 112) L (VL--CL)and H (VH--CH1)chain cDNAs and their deduced ORFs. The numbering underneath the aas follows the convention of Kabat et al. (1991). The positions of the FRs and the CDRs are highlighted. The 5' SacI and 3' XbaI sites used to clone the L chain and the 5' XhoI site used for the H chain are underlined. A naturally ocurring SpeI site in CDR-L1, which complicated the excision of gene III protein for expression of soluble Ab (K-B.G.S., G.Z. and A.E.K., data not shown), is also underlined. These sequences were deposited with GenBank under accession Nos. U04352 (481.1 H) and U04353 (481.1 L).
We used these sequences to prepare a computational model of the Fab 481.1 combining site and identified aa in CDRs L1, L3, H1, H2 and H3 that are likely to be involved in binding the herbicide. The model indicated diuron is bound by the following interactions: a hydrophobic pocket formed by Ala L96, Z y r H32'HS0,H100d and TrpH95, a hydrogen bond between His H35 and the carbonyl oxygen on diuron, and weak dipole interactions between the chlorine atoms on diuron and some combination of Gln TM, Gln L89, SerTM and Met m°°k of the Ab (Bell et al., 1995). This work was supported in part by the UC Toxic Substances Research and Teaching Program and a DOE Office of Technology Development subcontract LLLB244824 to A.E.K. K-B.G.S. was an NIH Postdoctoral Fellow (Grant AI-08710). A.E.K. is an investigator in the Environmental Health Sciences Center at UCB (N1EHS Grant ES01896). We thank Carlos Barbas (Scripps Research Institute) for providing pComb3 and pComb8,
and Dave Rockhold and Bill Belknap (USDA-ARS) for use of sequence analysis programs.
REFERENCES Barbas, C.F., Kang, A.S., Lerner, R.A. and Benkovic, S.J.: Assembly of combinatorial libraries on phage surfaces: The gene III site. Proc. Natl. Acad. Sci. USA 88 (1991) 7978-7982. Bell, C.W., Roberts, V.A., Scholthof, K.-B.G., Zhang, G. and Karu, A.E.: Recombinant antibodies to diuron: a model for the phenylurea binding site. In: Nelson, J.O., Karu, A.E. and Wong, R.B. (Eds.), Immunoanalysis of Agrochemicals: Emerging Technologies. American Chemical Society, Washington, DC, 1995, pp. 50-71. Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman, K.S. and Foeller, C.: Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, N.I.H., Washington, DC, 1991. Karu, A.E., Goodrow, M.H., Schmidt, D.J., Hammock, B.D. and Bigelow, M.W.: Synthesis of haptens and derivation of monoclonal antibodies for immunoassay of the phenylurea herbicide diuron. J. Agr. Food Chem. 42 (1994a) 301-309.
323
GENE 09246
Sequences of the cDNAs encoding the heavy- and light-chain Fab region of an antibody to the phenylurea herbicide diuron * (Recombinant DNA; complementarity determining region; antibody structure; phage display; mouse hybridoma)
C h r i s t o p h e r W. BelP, K a r e n - B e t h G. S c h o l t h o f
b,**, G u i s h e n g
Z h a n g T M a n d A l e x a n d e r E. K a r u a
"Hybridoma Facility, College of Natural Resources, University of California-Berkeley, Albany, CA 94706, USA; and bDepartment of Plant Biology, University of California-Berkelek; Berkeley, CA 94720, USA Received by C.M. Kane: 22 May 1995; Revised/Accepted: 30 June/5 July 1995; Received at publishers: 3 August 1995
SUMMARY
The cDNAs from a hybridoma (mAb 481.1) specific for diuron, a widely used phenylurea herbicide, were cloned into the phage display vector pComb8. Antigen-binding clones were selected by panning on diuron-hapten-BSA conjugates. The nucleotide and deduced amino-acid sequences encoding the Fab regions of the light (~:) and heavy (5') chains were determined. The light chain was from mouse ~cchain subgroup III and the heavy chain was a member of the mouse H chain subgroup III(d).
Diuron (3-(3,4-dichlorophenyl)-l,l-dimethylurea) is a widely used phenylurea herbicide. We have developed a series of mAb and EIAs that detect parts per billion (ng/ml) of diuron (Karu et al., 1994). We chose to clone the Ab-encoding genes from one of these hybridomas as the first step toward engineering new binding specificities for the phenylureas. We amplified the Fab genes from the cDNA ofmAb 481.1 (an IgG1K), and cloned them into Correspondence to: Dr. A.E. Karu, Hybridoma Facility, College of Natural Resources, University of California-Berkeley, 1050 San Pablo Ave, Albany, CA 94706, USA. Tel. (1-510) 643-7746; Fax (1-510) 642-0875; e-mail: [email protected] **Present addresses: (K-B.G.S.) Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA. Tel. (1-409) 845-8265; (G.Z.) Department of Transplantation Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. Tel. (1-412) 648-2080. * On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note. Abbreviations: aa, amino acid(s); Ab, antibody(ies); bp, base pair(s); BSA, bovine serum albumin; cDNA, DNA complementary to RNA; CDR, complementarity-determining region; C, constant region; EIA, enzyme immunoassay; Fab, antibody-binding fraction(s); FR, framework region; H, heavy; Ig, immunoglobulin; L, light; kb, kilobase(s) or 1000 bp; mAb, monoclonal Ab; nt, nucleotide(s); ORF, open reading frame.
SSDI 0378-1119(95~00531-5
the phage display vector pComb8 (Barbas et al., 1991). Antigen-binding phages were selected by rounds of panning with diuron hapten-BSA conjugates, and positive clones were subcloned into pComb3 (Barbas et al., 1991). Soluble recombinant Fab from these clones competitively bound diuron in EIAs with the same sensitivity and specificity as mAb 481.1 (K-B.G.S., G.Z. and A.E.K., data not shown). Sequences from one of the diuron-specific Fab 481.1 clones (112) are shown in Fig. 1. Each sequence encoded a single ORF with homology to known Ab aa sequences (Kabat et al., 1991). The L chain ORF extends from FR-1 (a SacI site) to the last residue (CysTM) of the CL region, and it is terminated by two in-frame stop codons (encoded within the primers). The H chain ORF begins at aa 6 (an XhoI site) in FR-1, the first five FR residues (AQVKL) being coded by the vector (Barbas et al., 1991 ), and continues to Cysnz35 at the end of Cm in the hinge region. From comparisons with the immunological database of Kabat et al. (1991), the H chain is a member of the mouse H chain subgroup III(d) and the L chain is a member of mouse ~ chain subgroup III. The hypervariable regions or CDRs, which form the antigen-binding loops of the antibody, were also identified (Fig. 1).
324
Fab 481.1 Lc (1<) i
FRI
C~R-LI
,
~R-2
GA$~T~;GTGATGACACAGTCTCCAGCT~GCTGTGTCTCTA~AGAGTGTCACCATCTCCTGCAGAGCCAGTGAAAGTGTTGAATATTATGGCACTAGTTTAATGCAGTC~GTACCAACAGA~%ACCAGGACAGCCACC~CTC
E
L
V
M
T
Q
I
S
P
A
S
L
CDR-L2
A
V
S
L
G
Q
S
V
T
I
S
C
R
A
I
C TCATCTATGGTGCATCCAACGTAGAATCTG la I Y G A S N V E S
S
E
S
V
E
Y
Y
27
a
b
c
d
e
G
T
S
L
M f 28 29 32 33
Q
W
Y
Q
Q
FR-3
K
P
G
150
Q
I
P
P
K
L
46
CDR-L3
G G G T C C C T G C C A G G T T T A G T G GCAGTGC4~ T C T G G G A C A G A C T T C A G C C T C A A C A T C C A T C C T G T G G A G G A G G A T G A T A T T G C A A T A T A T T T C T G T C A G ~GTAGGAAGGTTCCAG G V P A R F S G S G S G T D F S L N I H P V E E D D I A I Y F C Q Q S R K V P
CT A
300 96
C T C A G T C G T G T C47TT C T T G A A C A A C T T C T A C C C C A 2 ~ G A C A T C A A T G T C C F L N N F Y P K D I N V I 4 6
450
AAGTGGAAGATTGATGGCAG TGAACGACAAAAT GGCG TC CTGAACAG TTGGACTGATCAGGAC AGCAAAGACAGCAC CTACAG CA TGAGCAGCACC CT CACGTTGACCAAGGACGAGTATGAACGACATAACAGC TATAC CTGTGAGG CC K W K I D G S E R Q N G V L N S W T D Q D S K D S T Y S M S S T L T L T K D E Y E R H N S Y T C E A
600 196
FR4 I ACGTTCGGATCGGGGAC CAAGCTGGA.%ATAAAACGGGC TGATGCTGCAC CAAC TG TA TCCAT C TTC CCAC CATC CAGTGAGCAGTTAACATCTGGAGGTGC T F G S G T K L E I K R A D A A P T V S I F P P S S E Q L T S G G A S V V
CL
AC TCACAAGACATCAACTTCAC CCATCGTCAAGAGC TTCAACAGGAATGAGTGTTAATT CTAGA T H K T S T S P I V K S F N R N E C *
664 214
Fab 481.1 Hc (7) J~R1 . " CDR-~I "~ FR-2 I C TCGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTTGCATACATTAGTAGTGGC L
E
S
S
S
T
G
G
G
L
V
K
P
G
G
Y
A
D
T
V
K
G
CDR-H2 I
Y
S
L
K
L
S
C
A
A
S
G
F
T
F
S
D
R
F
T
I
S
R
D
N
A
K
N
T
L
F
L
I
Y
G
M
H
W
V
R
Q
A
P
E
K
G
L
E
W
V
M 82
T a
S b
L R c 83
S
E
D
T
A
M
Y
Y
C
A
R
FR-3 Q
A
"/
I
$ 52
S a
I
150 Ca
III W
mmm CDR-H3 I FR-4 I G G T C A T T A T T A T G T T A T G G A C T A C T C - G G G T C A A G G A - % CC T C a ~ G T C A C C G T C T C C T C A G C C A A A A C G A C A C C C C C A T C T G T C T A T C C A C T G G C C C CTC~?~ATC T G C T G C C C A A A C T A A C T C C A T G G T G A C C C T G G G A T G C C T G G ~ C A ~ G G G C G H Y Y V M D Y W G Q G T S V T V S S A K T T P P S V Y P L A P G S A A Q T N S M V T L G C L a
b
c
d
e
53
i00
450 146
k 101
CH-I TAT TTCCCTGAGCCAGTGACAGTGACcTGGAACTCTGGATCCcTGTCCAGcGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACcTCTACACTCTGAGCAGCTCAGTGACTGTCCCCT~CAGCACCTC,GCCCAGCcAGACCGTCACC Y F P E P V T V T W N S G S L S S G v H T F P A V L Q S D L ¥ T L S S S V T %" P S S T W P S Q T it
600 207
I TGCAACGTTGCCCAC CCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGC C N V A H P A S S T K V D K K I V
CCAGGGATTGTACTAGC P R D C T S
669
237
Fig. 1. The nt sequence of diuron-specific Fab 481.1 (clone 112) L (VL--CL)and H (VH--CH1)chain cDNAs and their deduced ORFs. The numbering underneath the aas follows the convention of Kabat et al. (1991). The positions of the FRs and the CDRs are highlighted. The 5' SacI and 3' XbaI sites used to clone the L chain and the 5' XhoI site used for the H chain are underlined. A naturally ocurring SpeI site in CDR-L1, which complicated the excision of gene III protein for expression of soluble Ab (K-B.G.S., G.Z. and A.E.K., data not shown), is also underlined. These sequences were deposited with GenBank under accession Nos. U04352 (481.1 H) and U04353 (481.1 L).
We used these sequences to prepare a computational model of the Fab 481.1 combining site and identified aa in CDRs L1, L3, H1, H2 and H3 that are likely to be involved in binding the herbicide. The model indicated diuron is bound by the following interactions: a hydrophobic pocket formed by Ala L96, Z y r H32'HS0,H100d and TrpH95, a hydrogen bond between His H35 and the carbonyl oxygen on diuron, and weak dipole interactions between the chlorine atoms on diuron and some combination of Gln TM, Gln L89, SerTM and Met m°°k of the Ab (Bell et al., 1995). This work was supported in part by the UC Toxic Substances Research and Teaching Program and a DOE Office of Technology Development subcontract LLLB244824 to A.E.K. K-B.G.S. was an NIH Postdoctoral Fellow (Grant AI-08710). A.E.K. is an investigator in the Environmental Health Sciences Center at UCB (N1EHS Grant ES01896). We thank Carlos Barbas (Scripps Research Institute) for providing pComb3 and pComb8,
and Dave Rockhold and Bill Belknap (USDA-ARS) for use of sequence analysis programs.
REFERENCES Barbas, C.F., Kang, A.S., Lerner, R.A. and Benkovic, S.J.: Assembly of combinatorial libraries on phage surfaces: The gene III site. Proc. Natl. Acad. Sci. USA 88 (1991) 7978-7982. Bell, C.W., Roberts, V.A., Scholthof, K.-B.G., Zhang, G. and Karu, A.E.: Recombinant antibodies to diuron: a model for the phenylurea binding site. In: Nelson, J.O., Karu, A.E. and Wong, R.B. (Eds.), Immunoanalysis of Agrochemicals: Emerging Technologies. American Chemical Society, Washington, DC, 1995, pp. 50-71. Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman, K.S. and Foeller, C.: Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, N.I.H., Washington, DC, 1991. Karu, A.E., Goodrow, M.H., Schmidt, D.J., Hammock, B.D. and Bigelow, M.W.: Synthesis of haptens and derivation of monoclonal antibodies for immunoassay of the phenylurea herbicide diuron. J. Agr. Food Chem. 42 (1994a) 301-309.