Obtaining and Purification of Recombinant Domain III of Human Alpha-Fetoprotein
| Autor: | D.A. Mazalev, M. D. Mollaev, E.D. Nikolskaya, M. V. Fomicheva, Mariia Mollaeva, Neonila V. Gorokhovets, Anna B. Pshenichnikova, Artur Zabolotskii, Maria Sokol |
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| Rok vydání: | 2021 |
| Předmět: | |
| Zdroj: | Biotekhnologiya. 37:32-42 |
| ISSN: | 2500-2341 0234-2758 |
| Popis: | Previous studies have demonstrated the applicability of alpha-fetoprotein or its receptor-binding domain fused to the 6-histidine tag at the C-terminus (rAFP3D-His6) as vectors for targeted delivery of antitumor agents. This tag is undesirable for further preclinical trials. Therefore, we designed a recombinant protein rAFP3D without any affine tags, and accessed its functional activity. The protein was produced as inclusion bodies in Escherichia coli BL21 (DE3) cells. Optimal conditions for washing inclusion bodies and refolding the target protein were selected. We used a saturated solution of (NH4)2SO4 as the primary purification step to precipitate the main fraction of protein admixtures. The second purification step included hydrophobic chromatography using butyl-cellulose. The identity of the protein sequence was confirmed by tandem mass spectrometry. Circular dichroism demonstrated the authenticity of the secondary structure. Fluorescently labeled rAFP3D actively penetrated into MCF-7 tumor cells. These results indicate that rAFP3D can be used for targeted drug delivery. Key words: alpha-fetoprotein, receptor-binding domain, recombinant protein, MALDI-MS, HPLC, drug delivery system Funding - The project was supported with Russian Foundation for Basic Research (project no. 18-29-09022/20). |
| Databáze: | OpenAIRE |
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