Advanced SearchSearch Tips
The development of anti-DR4 single-chain Fv (ScFv) antibody fused to Escherichia coli alkaline phosphatase
facebook(new window)  Pirnt(new window) E-mail(new window) Excel Download
 Title & Authors
The development of anti-DR4 single-chain Fv (ScFv) antibody fused to Escherichia coli alkaline phosphatase
Han, Seung Hee; Kim, Jin-Kyoo;
  PDF(new window)
Enzyme immunoassay to analyze specific binding activity of antibody to antigen uses horseradish peroxidase (HRP) or alkaline phosphatase (AP). Chemical methods are usually used for coupling of these enzymes to antibody, which is complicated and random cross-linking process. As results, it causes decreases or loss of functional activity of either antibody or enzyme. In addition, most enzyme assays use secondary antibody to detect antigen binding activity of primary antibody. Enzymes coupled to secondary antibody provide a binding signal by substrate-based color development, suggesting secondary antibody is required in enzyme immunoassay. Additional incubation time for binding of secondary antibody should also be necessary. More importantly, non-specific binding activity caused by secondary antibody should also be eliminated. In this study, we cloned AP isolated from Escherichia coli (E. coli) chromosome by PCR and fused to) hAY4 single-chain variable domain fragment (ScFv) specific to death receptor (DR4) which is a receptor for tumor necrosis factor related apoptosis induced ligand (TRAIL). hAY4 ScFv-AP expressed in E. coli showed 73.8 kDa as a monomer in SDS-PAGE. However, this fusion protein shown in size-exclusion chromatography (SEC) exhibited 147.6 kDa as a dimer confirming that natural dimerization of AP by non-covalent association induced ScFv-AP dimerization. In several immunoassay such as ELISA, Western blot and immunocytochemistry, it showed antigen binding activity by color development of substrates catalyzed by AP directly fused to primary hAY4 ScFv without secondary antibody. In summary, hAY4 ScFv-AP fusion protein was successfully purified as a soluble dimeric form in E. coli and showed antigen binding activity in several immunoassays without addition of secondary antibody which sometimes causes time-consuming, expensive and non-specific false binding.
alkaline phophatase;ELISA;immunocytochemistry;single-chain variable domain fragment (ScFv);Western blot;
 Cited by
Acchione, M., Kwon, H., Jochheim, C.M., and Atkins, W.M. 2012. Impact of linker and conjugation chemistry on antigen binding, fc receptor binding and thermal stability of model antibody-drug conjugates. MAbs. 4, 362-372. crossref(new window)

Carrier, A., Ducancel, F., Settiawan, N.B., Cattolico, L., Maillere, B., Leonetti, M., Drevet, P., Menez, A., and Boulain, J.C. 1995. Recombinant antibody-alkaline phosphatase conjugates for diagnosis of human iggs: Application to anti-hbsag detection. J. Immunol. Methods 181, 177-186. crossref(new window)

Coleman, J.E. 1992. Structure and mechanism of alkaline phosphatase. Ann. Rev. Biophy. Biomol. Struct. 21, 441-483. crossref(new window)

Ducancel, F., Gillet, D., Carrier, A., Lajeunesse, E., Menez, A., and Boulain, J.C. 1993. Recombinant colorimetric antibodies: construction and characterization of a bifunctional f (ab) 2/alkaline phosphatase conjugate produced in Escherichia coli. Biotechnology 11, 601-605. crossref(new window)

Duenas, M., Vazquez, J., Ayala, M., Soderlind, E., Ohlin, M., Perez, L., Borrebaeck, C., and Gavilondo, J. 1994. Intra-and extracellular expression of an scFv antibody fragment in E. coli: effect of bacterial strains and pathway engineering using GroES/L chaperonins. BioTechniques 16, 476-477, 480-473.

Harlow, E. and Lane, D. 1988. Antibodies: A lab manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

Hu, Z.Q., Li, H.P., Zhang, J.B., Huang, T., Liu, J.L., Xue, S., Wu, A.B., and Liao, Y.C. 2013. A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains. Anal. Chim. Acta. 764, 84-92. crossref(new window)

Kim, J.K., Tsen, M.F., Ghetie, V., and Ward, E.S. 1994. Identifying amino acid residues that influence plasma clearance of murine IgG1 fragments by site-directed mutagenesis. Eur. J. Immunol. 24, 542-548. crossref(new window)

Le Gall, F., Reusch, U., Little, M., and Kipriyanov, S.M. 2004. Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody. Protein Eng. Des. Sel. 17, 357-366. crossref(new window)

Lee, S.H., Park, D.W., Sung, E.S., Park, H.R., Kim, J.K., and Kim, Y.S. 2010. Humanization of an agonistic anti-death receptor 4 single chain variable fragment antibody and avidity-mediated enhancement of its cell death-inducing activity. Mol. Immunol. 47, 816-824. crossref(new window)

Lei, S.P., Lin, H., Wang, S.S., Callaway, J., and Wilcox, G. 1987. Characterization of the Erwinia carotovora pelB gene and its product pectate lyase. J. Bacteriol. 169, 4379-4383. crossref(new window)

Liu, X., Wang, H., Liang, Y., Yang, J., Zhang, H., Lei, H., Shen, Y., and Sun, Y. 2010. Production and characterization of a single-chain Fv antibody-alkaline phosphatase fusion protein specific for clenbuterol. Mol. Biotechnol. 45, 56-64. crossref(new window)

Mandecki, W., Shallcross, M.A., Sowadski, J., and Tomazic-Allen, S. 1991. Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat. Protein Eng. 4, 801-804. crossref(new window)

Muller, B.H., Chevrier, D., Boulain, J.C., and Guesdon, J.L. 1999. Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate for one-step immunodetection in molecular hybridization. J. Immunol. Methods 227, 177-185. crossref(new window)

Park, K.J., Park, D.W., Kim, C.H., Han, B.K., Park, T.S., Han, J.Y., Lillehoj, H.S., and Kim, J.K. 2005. Development and characterization of a recombinant chicken single-chain Fv antibody detecting Eimeria acervulina sporozoite antigen. Biotechnol. Lett. 27, 289-295. crossref(new window)

Sadeghi, H.M.M., Rabbani, M., and Fazeli, S. 2008. Optimization of alkaline phosphatase gene expression in E. coli. Res. Pharmaceut. Sci. 3, 35-39.

Solar, I. and Gershoni, J.M. 1995. Linker modification introduces useful molecular instability in a single chain antibody. Protein Eng. 8, 717-723. crossref(new window)

Suzuki, C., Ueda, H., Suzuki, E., and Nagamune, T. 1997. Construction, bacterial expression, and characterization of hapten-specific single-chain fv and alkaline phosphatase fusion protein. J. Biochem. 122, 322-329. crossref(new window)

Trinh, R., Gurbaxani, B., Morrison, S.L., and Seyfzadeh, M. 2004. Optimization of codon pair use within the (ggggs)3 linker sequence results in enhanced protein expression. Mol. Immunol. 40, 717-722. crossref(new window)

Volkel, T., Korn, T., Bach, M., Muller, R., and Kontermann, R.E. 2001. Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies. Protein Eng. 14, 815-823. crossref(new window)

Wang, C.L., Huang, M., Wesson, C.A., Birdsell, D.C., and Trumble, W.R. 1994. A single Fc binding domain-alkaline phosphatase gene fusion expresses a protein with both IgG binding ability and alkaline phosphatase enzymatic activity. Protein Eng. 7, 715-722. crossref(new window)

Wang, S., Zheng, C., Liu, Y., Zheng, H., and Wang, Z. 2008. Construction of multiform scFv antibodies using linker peptide. J. Genet. Genomics 35, 313-316. crossref(new window)

Wels, W., Harwerth, I.M., Zwickl, M., Hardman, N., Groner, B., and Hynes, N.E. 1992. Construction, bacterial expression and characterization of a bifunctional single-chain antibody-phosphatase fusion protein targeted to the human erbb-2 receptor. Biotechnology 10, 1128-1132. crossref(new window)

Whitlow, M., Bell, B.A., Feng, S.L., Filpula, D., Hardman, K.D., Hubert, S.L., Rollence, M.L., Wood, J.F., Schott, M.E., and Milenic, D.E. 1993. An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability. Protein Eng. 6, 989-995. crossref(new window)