Obtaining and Purifying the Recombinant Domain III of Human Alpha-Fetoprotein

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. The His6 tag is undesirable for further preclinical trials. Therefore, we designed a recombinant protein rAFP3D without any affine tags and assessed 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 (NH 4 ) 2 SO 4 solution at the primary purification stage to precipitate the main fraction of nontarget proteins. The second purification step included hydrophobic interaction 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 of the recombinant protein. MCF-7 tumor cells actively internalized fluorescently labeled rAFP3D. These results indicate that rAFP3D can be used for targeted drug delivery.