Optimization of the Expression of Recombinant Cetuximab Single-Chain Fragment Variable and Comparative its Purification with Magnetic Nanoparticles and Conventional Fast Protein Liquid Chromatography

Jalalvand, Masumeh and Falahzadeh, Khadijeh and Jalalvand, Amirmasoud and Mazloumi, Mohammadali and Shahsavari, Gholamreza (2023) Optimization of the Expression of Recombinant Cetuximab Single-Chain Fragment Variable and Comparative its Purification with Magnetic Nanoparticles and Conventional Fast Protein Liquid Chromatography. Biointerface Research in Applied Chemistry.

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Abstract

Various approaches are applied to purify recombinant proteins. Choosing an appropriate purification method seems necessary to achieve a high purity level. In the current study, we compared two different purification strategies, including nickel-nitrilotriacetic acid affinity chromatography using fast protein liquid chromatography system and Ni2+ -functionalized Fe3O4@polydopamine magnetic nanoparticles to purify recombinant cetuximab single-chain fragment variable which specifically attaches to epidermal growth factor receptor according to the affinity interactions between Ni2+ and a polyhistidine-tag on the carboxyl-terminus of cetuximab single-chain fragment variable. Epidermal growth factor receptor is overexpressed in many cancer types, especially colorectal cancer cells, making it a valuable candidate for targeted cancer therapy. We optimized expression conditions for recombinant cetuximab single-chain fragment variable in terms of isopropyl-L-thio-β-D-galactopyranoside concentration and cultivation temperature. The soluble cetuximab scFv was extracted from E. coli periplasm through osmotic shock. Ni2+ -functionalized Fe3O4@polydopamine magnetic nanoparticles were prepared using the co-precipitation method. Then, nickel-nitrilotriacetic acid affinity chromatography and Ni2+ -functionalized Fe3O4@polydopamine magnetic nanoparticles were employed to purify the cetuximab single-chain fragment variable. Although, according to our experiments, the main strength of nickel-nitrilotriacetic acid affinity chromatography is data reproducibility, this strategy has some big drawbacks, like a sophisticated, costly, and time-consuming purification process. Therefore, compared with nickel-nitrilotriacetic acid affinity chromatography using fast protein liquid chromatography, the Ni2+ -functionalized Fe3O4@polydopamine magnetic nanoparticles technique could be considered a simpler, faster and cheaper method for purification of desired recombinant proteins. Notably, the concentration of purified single-chain fragment variable using Ni2+ - functionalized Fe3O4@polydopamine magnetic nanoparticles gradually decreased in the subsequent batch reactions

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