Antibody Expression and Engineering - American Chemical Society

Chapter 11

Strong and Specific Peroxidase Activity with an Antibody L Chain-Porphyrin Fe(III) Complex Tadayuki Imanaka and Masahiro Takagi

Downloaded by UNIV LAVAL on October 6, 2015 | http://pubs.acs.org Publication Date: August 18, 1995 | doi: 10.1021/bk-1995-0604.ch011

Department of Biotechnology, Faculty of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565, Japan

We attempt to generate monoclonal antibodies as host proteins for the functional molecular porphyrin, and thus engineer a new type of catalytic antibody. TCPP (meso-tetrakis (4-carboxyphenyl) porphyin) was chemically synthesized and Balb/c mice were immunized using TCPP as a hapten. Two hybridoma cells (03-1, 13-1), which produce monoclonal antibody against TCPP, were obtained. One of the monoclonal antibodies, Mab 03-1, exhibited enhanced peroxidase activity when TCPP Fe(III) was incorporated. Genes for both H and L chains of monoclonal antibodies were cloned, sequenced and overexpressed using E. coli as a host. ELISA and fluorescence quenching method show that the antibody L chains from both Mab 031 and Mab 13-1 have specific strong interaction with TCPP. Furthermore, a TCPP Fe(III) complex with the L chain from Mab 13-1 exhibits much higher peroxidase activity than TCPP Fe(III) alone. The enzyme activity was detectable with pyrogallol and ABTS(2,2-azinobis 3-ethylbenzthiazolin-6-sulfonic acid) but not with catechol. This result provides a new concept of catalytic antibody using monoclonal antibody or antibody fragment as a host carrier protein for a functional molecule.

Antibodies have been demonstrated to catalyze a wide variety of transformations such as ester hydrolysis (7-5), pericyclic(4,) photo-chemical (5j, ligand substitution^) and redox reactions(7.) These new antibodies were designated as catalytic antibodies or abzymes. Even though several strategies have been reported to generate these catalytic antibodies, we attempt here to engineer a new way to generate a new catalytic antibody with peroxidase activity using a monoclonal antibody as the host carrier protein for a functional molecule, porphyrin (Figure 1). We choose porphyrin because many active centers of various peroxidases and catalases consist of a porphyrin molecule encapsulated by a protein molecule. Previous construction of catalytic antibody used N-methylmeso-porphyrin IX, a presumed transition-state analogue for porphyrin metalation as a hapten. This antibody-porphyrin complex was formed to catalyze the reduction of hydrogen peroxide by several typical

0097H5156/95/0604-0138$12.00/0 1995 American Chemical Society

In Antibody Expression and Engineering; Wang, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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139

chromogenic peroxidae substates(8). We synthesized a TCPP which exhibits higher substrate specificity than N-methylmeso-porphyrin DC. Furthermore, recombinant antibody L chain was found to have specific and strong interaction with porphyrin. The L chain complex exhibited even higher peroxidase activity than the complex of the entire antibody and porphyrin.


Results and Discussion Raising antibody against porphyrin. TCPP was covalently attached to the carrier protein, keyhole limpet hemocyanin (KLH) using water-soluble carbodiimide, l-(3dimethylamino)propyl-3-ethyl-carbodiimide, or carbonyl diimidazol (Figure 2). The conjugates were then purified by column chromatography on Sephadex G-50. Balb/c mice were immunized with the K L H conjugate emulsified in complete Freund's adjuvant. Cell fusion was carried out using p3x63-Ag8.653 myeloma as the fusion partner and polyethylene glycol as die fusion reagent. Two hybridoma cells (03-1, 13-1) were screened and propagated in ascites as described previously(9;. Two monoclonal antibodies from hybridoma cells (Mab 03-1, Mab 13-1) were purified with a protein A column. Specific interaction of Mab 03-1 and Mab 13-1 with TCPP was examined by ELISA (enzyme-linked immunosorbent assay) and purified monoclonal antibodies were used for further studies. Gene cloning and expression of monoclonal antibody in Escherichia coli. Hybridoma cells, 03-1 and 13-1, were cultivated and cellular mRNA was purified by Quick Prep mRNA Purification kit (Pharmacia P-L Biochemicals Inc., Milwaukee, WI.). Isolated mRNA was then used to construct cDNA using an oligo(dT)18 primer and reverse transcriptase. Genes for both L and H chains of two monoclonal antibodies (Mab 03-1, Mab 13-1) were amplified using PCR (polymerase chain reaction) and cloned into Immunozap L and H vectors(70) respectively. Nucleotide sequences of the cloned DNA fragments encoding L and H chains were determined by dideoxy chain termination method(/7 ) (GenBank Acc. No. D29667-D29670). Deduced amino acid sequences were then compared between Mab 03-1 and Mab 13-1 antibodies. The amino acid sequences of the two antibodies were found to be different only in hyper variable regions (CDR 1,2 and 3). Therefore, Mab 03-1 and Mab 13-1 most likely recognize porphyrin in a different manner. Induced expression of cloned monoclonal antibody genes was then attempted. However, the expression level of genes cloned into Immunozap vectors was very low. Therefore, genes for the appropriate L and H chain s were subcloned respectively into an expression vector, pET8-c, which has a strong promoter sequence for T7RNA polymerase(/2.) Overexpression of antibody protein was successfully performed using E. coli BL21(DE3) as a host. Antibody proteins could be recovered as inclusion bodies in insoluble cytoplasmic fraction. Inclusion bodies were purified and solubilized using 6 M guanidine hydrochloride. Then, the purified proteins were refolded by dialysis. The homogeneity of purified antibody L and H chains was examined by SDS polyacrylamide gel electrophoresis. Dissociation constants of monoclonal antibodies Specific binding between each recombinant antibody chain and porphyrin (TCPP) was examined by ELISA using anti-Mab 03-1 and anti-Mab 13-1 as secondary antibodies. H chains from both monoclonal antibodies did not show antigen binding, while specific binding between independent antibody L chain and TCPP


140

ANTIBODY EXPRESSION AND ENGINEERING

Ferric Ion Substrate

Product

Substrate 4.

Peroxidase

,

HzO


Hapten (Porphyrin)

(U)J

Antigen-Antibody Interaction New Catalytic Antibody

Antibody (Host Protein) No BindingN^No Activity

Figure 1. Illustration of a concept for the new catalytic antibody.

TCPP-KLH

TCPP TCPP KLH

: meso-tetrakis (4-carboxyphenyl) porphyrin keyhole limpet hemocyanin Figure 2. Structure of TCPP


11.

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was observed for both 03-1 and 13-1 antibodies (data not shown). Kinetics of binding was more quantitalively analyzed. Using fluorescence quenching method, dissociation constants (Kd) for Mab 03-1, Mab 13-1 and two L chains were determined(;j) (Table I). Interestingly, L chains of both 03-1 and 13-1 monoclonal antibodies have specific but slighdy weaker interaction with TCPP than Mab 03-1 and Mab 13-1. Moreover, Mab 13-1 did not show any specific interaction against TCPP Fe(III). Independent L chain from Mab 13-1 exhibits specific interaction (Kd=1.4 χ 10-5) with TCPP Fe(III).


Table I: Dissociation constants (Kd) for TCPP-antibody complex (M)

Antibody Protein

ICEE

Mab 03-1 L03-1 Mab 13-1 L13-1

6.4 x l O 2.4xl0l.OxlO" ^ 2.6 x l O -

TCPPffcOn)) 8

6

7

6

7

1.5 χ 10" 1.0 χ 10' " ) 1.4xl0-

5

b

5

a) Kd was determined by ELISA. Other Kd values were by fluorescence quenching. b) No specific interaction was observed. Some naturally occurring active antibodies of a camel are composed of heavy chain dimmers and devoid of L chain(/4;. It was also reported that recombinant single immunoglobulin variable domains of Η chain secreted from E. coli had specific binding activities(75J. However, specific antigen binding using isolated L chain has not been reported before. Measurement of peroxidase activity Since L chains of the Mab 03-1 and Mab 13-1 antibodies exhibit specific interactions with TCPP Fe(III), we are interested in examining whether a peroxidase reaction can be enhanced using the complex of L chain and TCPP Fe(III). Using pyrogallol as a substrate, peroxidase activity was tested for Mab 031 and Mab 13-1 L chain complexes with TCPP Fe(IH) and compared with those of TCPP Fe(ffl) alone and Mab 03-1-TCPP Fe(ffl) (Figure 3a.) The L chain of Mab 03-1 exhibits slighdy higher activity than TCPP Fe(III) but the activity was still lower than that of Mab 03-1. Interestingly, the 13-1 L chain exhibited about 3.5 times higher peroxidase activity than Mab 03-1 (Figure 3a). These results and the observation that Mab 13-1 did not bind to TCPP Fe(III) suggested that Η chain of Mab 13-1 antibody might interfere with the binding of TCPP Fe(III). This resultant complex of 13-1 antibody L chain and TCPP Fe(III) exhibited the highest peroxidase activity among all the tested and previously reported catalytic antibodies. Therefore, we named these chains of antibody fragments with peroxidase activity L-zyme. Specificity of the peroxidase activity was also examined using other substrates, ABTS, (2,2-azinobis 3-ethylbenz-thiazolin-6-sulfonic acid) (Figure 3b) and catechol. A significant increase in peroxidase activity for ABTS was observed using the complex of 13-1 L chain and TCPP Fe(III) (Figure3b). Yet, no activity was detected when catechol was used as a substrate (data not shown). The


142

ANTIBODY EXPRESSION AND ENGINEERING

(A) Pyrogallol

(B) ABTS


H o J J ? OH

H o 5 j D

H CM CM

CM

CM

• ··· Ι·β o

tAîîïïïïîIÏ"

6Ç

0

100 J00 300 400 500

T1ME(MC)

Figure 3. Reaction of pyrogallol (A) and ABTS (B). Peroxidase reaction by complexes of antibody proteins and TCPP Fe(III). Symbols • Δ • Ο and · indicate TCPP Fe(III) mixed with none, BSA, Mab 03-1,03-1 L chain, and 13-1 L chain, respectively. The reaction was performed at 37° C as follows. Reaction mixture containing 5.0 χ 10-7 M TCPP Fe(III), 8.0 χ 10-7 M antibody protein, 1.2 χ 10-3 M substrate, and 5.0 χ 10-3 hydrogen peroxide dissolved into TAB buffer (90 mM Tris-acetate pH 8.0) was prepared and incubated at 37° C. Absorban ce of wavelength at 420 nm (pyrogallol) or 414 nm (ABTS) was monitored by Shimadzu UV160 UV-visible resording spectrophotometer. The reaction mixtures without antibody protein and with BSA (bovine serum albumin) at the same concentration were prepared as control.


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enhancement of peroxidase activity for ABTS might be based on a similar mechanism as explained above for pyrogallol. However, the intensity of enhancement was found to be smaller probably due to the larger size of ABTS (Figure 3b).


Conclusions We have demonstrated that peroxidase activity could be engineered by the formation of chemically synthesized TCPP Fe(III) and anti-TCPP monoclonal antibody complex. This method provides a new approach to develop a catalytic antibody. Furthermore, it was shown that recombinant L chain could recognize and interact with TCPP and TCPP Fe(III). The complex of 13-1 L chain and TCPP Fe(III) exhibits much higher peroxidase activity than TCPP Fe(III) alone or Mab 03-1 with TCPP Fe(III). We are now examining the molecular mechanism of this new L-zyme through amino acid substitutions and structural studies. Literature Cited 1. A. Tramontano, K.D. Janda, R.A. Lerner, Science, 234, 1566 (1986). 2. S.J. Pollack, J.W. Jacobs, P.G. Schultz, Science, 234, 1570 (1986). 3. J.W. Jacobs, P.G. Schultz, R. Sugasawara, J. Powell, J. Am.Chem.Soc., 109, 2174 (1987). 4. A.C. Braisted and P.G. Schultz, J. Am. Chem. Soc. 112, 7430, (1990). 5. A.G. Cochran, R. Sugasawara, P.G. Schultz, J. Am. Chem. Soc. 110, 7888 (1988). 6. K.M. Shokat, C.J. Leumann, R. Sugasawara, P.G. Shcultz, Nature. 338, 269 (1989). 7. N. Janjic, a. Tramontano, J. Am.Chem.Soc. 111, 9101 (1989). 8. A.G. Cochran and P.G. Schultz, J. Am. Chem. Soc. 112, 9414 (1990). 9. A. Harada, K. Okamoto, M. Kamachi, Chem. Lett. 953 (1991). 10. R.L. Mullinax et al. Proc. Natl. Acad. Sci. USA. 87, 8095 (1990). 11. F. Sanger, S. Nicklen, A.R. Coulson, Proc. Natl. Acad. Sci. USA. 74, 5463 (1977). 12. F.W. Studier, A.H. rosenberg, J.J. Dunn, J.W. Dubendorff, Methods Enzymol. 185, 60 (1990). 13. G. Scatchard, Ann. N.Y. Acad. Sci. 51, 660 (1949). 14. C. Harmers-Casterman et al., Nature, 363, 446 (1993). 15. E.S. Ward, d. Gussow, A.D. Griffiths, P.T. Jones, G. Winter, Nature 341, 544546 (1989). R E C E I V E D June 13, 1995


Antibody Expression and Engineering - American Chemical Society

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