Extraction and purification of recombinant single chain antibody recognizing blood antigens
Keywords:Escherichia coli, antiA-scFv, blood type A, purification, single chain antibody.
In our previous study, we reported the expression of a recombinant single chain fragment variable (scFv) antibody that recognized blood type A antigen (antiA-scFv) in E. coli. When when it was expressed as it is alone, antiA-scFv was produced as inclusion body. In contrast, SM/antiA-scFv was synthesized in soluble form when it was fused to small ubiquitin modifier (SUMO). Here, we present the extraction and purification of antiA-scFv in the inclusion body as well as in the soluble form and evaluate the antiA-scFv antibody activity. The results show that only fusion expression of soluble SM/antiA-scFv has biological activity of the antibody. SM/antiA-scFv was separated by fractional precipitation with 20% ammonium sulfate, and then washed with buffers to collect the pure antiA-scFv with SUMOprotease treatment. The purity of recombinant antibody was 89% and the yield of 64.9 mg/L of bacterial culture. The antibody has a polymer structure and could bind to purified antigen as well as agglutinate with red blood cell, but the specificity of the antibody was not good enough for the antigen and red blood cell of blood type A. This is the first report in Vietnam showing the extraction and purification of the recombinant single chain antibody recognizing antigen of ABO system using E. coli expression system. It can be considered as a reference for further studies to improve the specificity of recombinant antibody antiA-scFv to identify ABO-type blood antigens.
Ahmad Z. A., Yeap S. K., Ali A. M., Ho W. Y., Alitheen N. B. M., Hamid M., 2012. ScFv antibody: Principles and clinical application. Clin. Dev. Immunol., 2012: 1–15.
Bothmann H., Plückthun A., 2000. The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA: I. Increased functional expression of antibody fragments with and without cis-prolines. Journal of Biological Chemistry, 275(22): 17100–17105.
Bu D., Zhou Y., Tang J, Jing, F., Zhang, W., 2013. Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli. Protein Expr. Purif., 92 (2013): 203–207.
Dang T. N. H., Le T. T. H., Truong N. H., 2018. Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with SUMO and chaperones in Escherichia coli. Academia Journal of Biology, 40(4): 103–110.
Dang Thi Ngoc Ha, Nguyen Thi Trung, Le Thi Thu Hong, Do Thi Huyen, Truong Nam Hai, 2017. Selection of fermentation condition for expression of recombinant single chain antibody recognizing the antigen of blood type A in Escherichia coli. Tap chi Sinh hoc, 39(2): 212-222. http://doi.org/625/0866-7160/v39n2.8955.
Frenzel A., Hust M., Schirrmann T., 2013. Expression of recombinant antibodies. Frontiers in Immunology, 4 (217): 1–20.
Friedrich L., Stangl S., Hahne H., Küster B., Köhler P., Multhoff G., Skerra A., 2010. Bacterial production and functional characterization of the Fab fragment of the murine IgG1/λ monoclonal antibody cmHsp70.1, a reagent for tumour diagnostics. Protein Engineering, Design and Selection, 23(4): 161–168.
Furuta M., Uchikawa M., Ueda Y., Yabe T., Taima T., Tsumoto K., Kojima S., Juji T., Kumagai I., 1998. Construction of mono- and bivalent human single-chain Fv fragments against the D antigen in the Rh blood group: multimerization effect on cell agglutination and application to blood typing. Protein engineering, 11: 233–241.
Golchin M., Khalili-Yazdi A., Karamouzian M., Abareghi A. 2012. Latex agglutination test based on single-chain Fv recombinant antibody fragment. Scand. J. Immunol., 75: 38–45.
Laemmli U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680–685.
Proba K., Wörn A., Honegger A., Plückthun A., 1998. Antibody scFv fragments without disulfide bonds, made by molecular evolution. Journal of Molecular Biology, 275(2): 245–253.
Skerra A., Plückthun A., 1988. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science (New York, N.Y.), 240: 1038–1041.
Song H. N., Jang J. H., Kim Y. W., Kim D. H., Park S. G., Lee M. K., Paek S. H., Woo E. J., 2014. Refolded scFv antibody fragment against myoglobin shows rapid reaction kinetics. International Journal of Molecular Sciences, 15(12): 23658–23671.
Spadiut O., Capone S., Krainer F., Glieder A., Herwig C., 2014. Microbials for the production of monoclonal antibodies and antibody fragments. Trends in Biotechnology, 32(1): 54–60.
Wörn A., Der Maur A. A., Escher D., Honegger A., Barberis A., Plückthun A., 2000. Correlation between in vitro stability and in vivo performance of anti- GCN4 intrabodies as cytoplasmic inhibitors. Journal of Biological Chemistry, 275(4): 2795–2803.
Wu S., Ke A., Doudna J. A., 2007. A fast and efficient procedure to produce scFvs specific for large macromolecular complexes. J. Immunol. Methods. 318(1–2): 95–101.