Abstract

    Open Access Research Article Article ID: JBM-3-107

    Different Strategies used in the Production of human monoclonal scfv Antibodies Specific to Dimers of Membrane Receptors

    Sylwia Łukasiewicz*, Agata Stachowicz, Ewelina Fic, Ewa Błasiak, Agata Kowalik and Marta Dziedzicka-Wasylewska

    Phage display technology is one of the methods widely adopted for the production of human monoclonal antibodies. The subject of the present research was the scFv - single chain variable fragment antibody - which was able to recognize the D2-5-HT1A heteromer formed by dopamine D2 and serotonin 5-HT1A receptors. The antibody was selected using the phage display technique and the human antibody scFv phagemid library Tomlinson J. We investigated two expression systems: prokaryotic and eukaryotic. The results indicate that both of these systems (E.coli HB2151 and High Five cells (BTI-TN-5B1-4) of Trichopulsia ni) are suitable for the production of scFv antibodies, but the scFvs obtained from insect cells could attain a higher protein concentration. Next, we examined a few purification methods: immobilized-metal affinity chromatography (IMAC), affinity chromatography with the c-myc tag and the methods using protein L or A agarose, which are applicable to scFvs lacking tags. In the bacterial as well as insect cell expression systems, protein L agarose chromatography gave satisfactory results, and in our opinion, it is the best choice as a purification resin. Additionally, circular dichroism measurements confirmed the predominant content of secondary structures in the tested antibodies, which is consistent with the literature data.

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    Published on: Apr 19, 2019 Pages: 14-20

    Full Text PDF Full Text HTML DOI: 10.17352/jbm.000007
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