Biopolym. Cell. 2009; 25(3):245-249.
http://dx.doi.org/10.7124/bc.0007E3
Short Communications
Construction of bifunctional molecules specific to antigen and antibody’s Fc-fragment by fusion of scFv-antibodies with staphylococcal protein A
1Oliinyk O. S., 1Kaberniuk A. A., 1Redchuk T. A., 1Kolibo D. V., 1Komisarenko S. V.
  1. Palladin Institute of Biochemistry, NAS of Ukraine
    9, Leontovycha Str., Kyiv, Ukraine, 01601

Abstract

Aim. To develop approach for detection of scFv and their complexes with antigens. Methods. The fusion proteins, which include sequences of scFv and staphylococcal protein A, were constructed and the obtained bifunctional molecules were immunochemically analysed. Results. It was shown, that scFv fused with protein A and their complexes with antigens are effectively recognized by labelled immunoglobulins with unrestricted antigenic specificity. Conclusions. The fusion of scFv with protein A fragment is a perspective approach to increase the efficiency of application in ELISA. The obtained scFv, fused with protein A, could be used for development of test-systems for the detection of diphtheria toxin.
Keywords: scFv, diphtheria toxin, bifunctional molecules
Full text: (PDF, in Ukrainian)

References

[1] Holliger Ph., Hudson P. J. Engineered antibody fragments and the rise of single domains Nat. Biotechnol 2005 23, N 9:1126–1136.
[2] Wang S. H., Zhang J. B., Zhang Z., P. Zhou Y. F., Yang R. F., Chen J., Guo Y. C., You F., Zhang X. E. Construction of single chain variable fragment (ScFv) and BiscFv-alkaline phosphatase fusion protein for detection of Bacillus anthracis Anal. Chem 2006 78, N 4:997–1004.
[3] Rau D., Kramer K., Hock B. Single-chain Fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for ELISA J. Immunoas. Immunochem 2002 23, N 2:129–143.
[4] Aubrey N., Devaux C., di Luccio E., Goyffon M., Rochat H., Billiald P. A recombinant scFv/streptavidin-binding peptide fusion protein for the quantitative determination of the scorpion venom neurotoxin AahI Biol. Chem 2001 382, N 11:1621–1628.
[5] Kipriyanov S. M., Breitling F., Little M., Dubel S. Singlechain antibody streptavidin fusions: tetrameric bifunctional scFv-complexes with biotin binding activity and enhanced affinity to antigen Hum. Antibodies Hybridomas 1995 6, N 3:93–101.
[6] Braren I., Greunke K., Umland O., Deckers S., Bredehorst R., Spillner E. Comparative expression of different antibody formats in mammalian cells and Pichia pastoris Biotechnol. Appl. Biochem 2007 47, N 4:205–214.
[7] Cao M., Cao P., Yan H., Ren F., Lu W., Hu Y., Zhang S. Construction and characterization of an enhanced GFP-tagged anti-BAFF scFv antibody Appl. Microbiol. Biotechnol 2008 79, N 3:423–431.
[8] Hobera S., Nordb K., Linhultc M. Protein A chromatography for antibody purification J. Chromatogr. B 2007 848, N 1:40–47.
[9] Tai M. S., Mudgett-Hunter M., Levinson D., Wu G. M., Haber E., Oppermann H., Huston J. S. A bifunctional fusion protein containing Fc-binding fragment B of staphylococcal protein A amino terminal to antidigoxin single-chain Fv Biochemistry 1990 29, N 35:8024–8030.
[10] Gandecha A., Owen M. R., Cockburn W., Whitelam G. C. Antigen detection using recombinant, bifunctional single-chain Fv fusion proteins synthesised in Escherichia coli Protein Exp. Purif 1994 5, N 4:385–390.
[11] Sambrook J., Fritsch E. F. Molecular cloning: A laboratory manual New York: Cold Spring Harbor Lab., 2001 999 p.
[12] Oliinyk O. S., Kaberniuk A. A., Redchuk T. A., Korotkevich N. V., Labyntsev A. U., Romanyuk S. I., Kolibo D. V., Komisarenko S. V. Construction of immune library of murine immunoglobulin genes and screening of single chaine Fv-antibodies to diphtheria toxin B subunit. Ukr. Biokhim. Zh. 2009; 81(2):68–79.
[13] Schagger H., von Jagow G. Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa Anal. Biochem 1987 166:368–379.
[14] Kabernyuk AA, Oliinyk OS, Redchuk TA, Romanyuk SI, Kolibo DV, Komisarenko SV. The cloning of genes of diphtheria toxin's recombinant subunits of Corynebacterium diphtheriae and their expression in Esherichia coli cells. Dopovidi Nats Akad Nauk Ukrainy. 2008; (3):160-6.
[15] Inouye S., Sahara Y. Soluble protein expression in E. coli cells using IgG-binding domain of protein A as a solubilizing partner in the cold induced system Biochem. and Biophys. Res. Communs 2008 376, N 3:448–453.
[16] Verdoliva A., Pannone F., Rossi M., Catello S., Manfredi V. Affinity purification of polyclonal antibodies using a new allD synthetic peptide ligand: comparison with protein A and protein G J. Immunol. Meth 2002 271, N 1–2:77–88.