Browse > Article
http://dx.doi.org/10.4014/jmb.1010.10022

Production of a Recombinant Anti-Human CD4 Single-Chain Variable-Fragment Antibody Using Phage Display Technology and Its Expression in Escherichia coli  

Babaei, Arash (Department of Biology, Faculty of Sciences, University of Malayer)
Zarkesh-Esfahani, Sayyed Hamid (Department of Biology, Faculty of Sciences, University of Isfahan)
Gharagozloo, Marjan (Department of Immunology, School of Medicine, Isfahan University of Medical Sciences)
Publication Information
Journal of Microbiology and Biotechnology / v.21, no.5, 2011 , pp. 529-535 More about this Journal
Abstract
Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications.
Keywords
ScFv; anti-CD4; phage display; recombinant antibody;
Citations & Related Records

Times Cited By Web Of Science : 1  (Related Records In Web of Science)
연도 인용수 순위
  • Reference
1 Arbabi-Ghahroudi, M., J. Tanha, and R. MacKenzie. 2005. Prokaryotic expression of antibodies. Cancer Metastasis Rev. 24: 501-519.   DOI   ScienceOn
2 Benjamin, R. J. and H. Waldmann. 1986. Induction of tolerance by monoclonal antibody therapy. Nature 320: 449-451.   DOI   ScienceOn
3 Bird, R. E., K. D. Hardman, J. W. Jacobson, S. Johnson, B. M. Kaufman, and S. M. Lee. 1988. Single-chain antigen-binding proteins. Science 242: 423-426.   DOI
4 Peitsch, M. C. and N. Guex. 1997. Swiss-Model and the Swiss- PDBViewer: An environment for comparative protein modeling. Electrophoresis 18: 2714-2723.   DOI   ScienceOn
5 Peterson, A. and B. Seed. 1988. Genetic analysis of monoclonal antibody and HIV binding sites on the human lymphocyte antigen CD4. Cell 54: 65-72.
6 Qiagen. 2001. QIAgen Detection and Assay Handbook. Qiagen, Hilden, Germany.
7 Qin, S. X., S. Cobbold, R. Benjamin, and H. Waldmann. 1989. Induction of classical transplantation tolerance in the adult. J. Exp. Med. 169: 779.   DOI   ScienceOn
8 Saerens, D., F. Frederix, G. Reekmans, K. Conrath, K. Jans, L. Brys, et al. 2005. Engineering camel single-domain antibodies and immobilization chemistry for human prostate-specific antigen sensing. Anal. Chem. 77: 7547-7555.   DOI   ScienceOn
9 Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
10 Schwede, T., J. Kopp, N. Guex, and M. C. Peitsch. 2003. SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res. 31: 3381.   DOI
11 Skov, L., K. Kragballe, C. Zachariae, E. R. Obitz, E. A. Holm, G. B. E. Jemec, et al. 2003. HuMax-CD4: A fully human monoclonal anti-CD4 antibody for the treatment of psoriasis vulgaris. Arch. Dermatol. 139: 1433.   DOI   ScienceOn
12 Cabilly, S. 1989. Growth at sub-optimal temperatures allows the production of functional, antigen-binding Fab fragments in Escherichia coli. Gene 85: 553-557.   DOI   ScienceOn
13 Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.   DOI   ScienceOn
14 Burastero, S. E., M. Figini, B. Frigerio, P. Lusso, L. Mollica, and L. Lopalco. 2009. Protective versus pathogenic anti-CD 4 immunity: Insights from the study of natural resistance to HIV infection. J. Trans. Med. 7: 101.   DOI
15 Burkly, L. C., D. Olson, R. Shapiro, G. Winkler, J. J. Rosa, D. W. Thomas, C. Williams, and P. Chisholm. 1992. Inhibition of HIV infection by a novel CD4 domain 2-specific monoclonal antibody. Dissecting the basis for its inhibitory effect on HIVinduced cell fusion. J. Immunol. 149: 1779.
16 Glockshuber, R., M. Malia, I. Pfitzinger, and A. Plückthun. 1990. A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29: 1362.   DOI   ScienceOn
17 Carmen, S. and L. Jermutus. 2002. Concepts in antibody phage display. Brief. Funct. Genomics 1: 189.   DOI   ScienceOn
18 Carrel, S., A. Moretta, G. Pantaleo, G. Tambussi, P. Isler, B. Perussia, and J. C. Cerottini. 1988. Stimulation and proliferation of CD4+ peripheral blood T lymphocytes induced by an anti- CD4 monoclonal antibody. Eur. J. Immunol. 18: 333-339.   DOI   ScienceOn
19 Chester, K. A., R. H. J. Begent, L. Robson, P. A. Keep, R. B. Pedley, J. A. Boden Libiol, et al. 1994. Phage libraries for generation of clinically useful antibodies. The Lancet 343: 455- 456.   DOI   ScienceOn
20 Gutstein, N. L., W. E. Seaman, J. H. Scott, and D. Wofsy. 1986. Induction of immune tolerance by administration of monoclonal antibody to L3T4. J. Immunol. 137: 1127.
21 Hasler, P. 2006. Biological therapies directed against cells in autoimmune disease Springer Seminars in Immunopathology 27: 443-456.
22 Huston, J. S., D. Levinson, M. Mudgett-Hunter, M. S. Tai, J. Novotny, and M. N. Margolies. 1988. Protein engineering of antibody binding sites: Recovery of specific activity in an antidigoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85: 5879-5883.   DOI   ScienceOn
23 McCafferty, J., H. Hoogenboom, and D. Chiswell. 1996. Antibody Engineering: A Practical Approach. Oxford University Press, USA.
24 Jabado, N., A. Pallier, F. Le Deist, F. Bernard, A. Fischer, and C. Hivroz. 1997. CD4 ligands inhibit the formation of multifunctional transduction complexes involved in T cell activation. J. Immunol. 158: 94.
25 Jameson, B. A., P. E. Rao, L. I. Kong, B. H. Hahn, G. M. Shaw, L. E. Hood, and S. B. Kent. 1988. Location and chemical synthesis of a binding site for HIV-1 on the CD4 protein. Science 240: 1335.   DOI
26 Janeway, C. A., P. Travers, M. Walport, and M. Shlomchik. 2005. Immunobiology: The Immune System in Health and Disease. Garland Science Publishing, New York, USA.
27 Kramer, K. and B. Hock. 2003. Recombinant antibodies for environmental analysis. Anal. Bioanal. Chem. 377: 417-426.   DOI   ScienceOn
28 Laemmli, U. K. 1970. Most commonly used discontinuous buffer system for SDS electrophoresis. Nature 227: 680-685.   DOI   ScienceOn
29 Morgenthaler, N. G., K. Hodak, J. Seissler, H. Steinbrenner, I. N. A. Pampel, M. Gupta, A. M. McGregor, W. A. Scherbaum, and J. P. Banga 1999. Direct binding of thyrotropin receptor autoantibody to in vitro translated thyrotropin receptor: A comparison to radioreceptor assay and thyroid stimulating bioassay. Thyroid 9: 467-475.   DOI
30 Peitsch, M. C. 1995. Protein modeling by e-mail. From amino acid sequence to protein structure: A free one-hour service. Nature Biotechnol. 13: 658-698.   DOI   ScienceOn
31 Whitlow, M. and D. Filpula. 1991. Single-chain Fv proteins and their fusion proteins. Methods 2: 97-105.   DOI
32 Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350.   DOI   ScienceOn
33 Truneh, A., D. Buck, D. R. Cassatt, R. Juszczak, S. Kassis, S. E. Ryu, D. Healey, R. Sweet, and Q. Sattentau. 1991. A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion. J. Biol. Chem. 266: 5942.
34 Tsygankov, A. Y., B. M. Broker, A. H. Guse, U. Meinke, E. Roth, C. Rossmann, and F. Emmrich. 1993. Preincubation with anti-CD4 influences activation of human T cells by subsequent co-cross-linking of CD4 with CD3. J. Leukocyte Biol. 54: 430.
35 Wingren, C. and C. A. K. Borrebaeck. 2006. Antibody microarrays: Current status and key technological advances. OMICS 10: 411-427.   DOI   ScienceOn
36 Yang, J., R. Chen, J. Wei, F. Zhang, Y. Zhang, L. Jia, et al. Production and characterization of a recombinant single-chain antibody against Hantaan virus envelop glycoprotein. Appl. Microbiol. Biotechnol. 86: 1067-1075.