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



Jalalvand M¹ ; Falahzadeh K¹ ; Jalalvand A² ; Mazloumi M¹ ; Bakhshandeh H³ ; Shahsavari G⁴ ; Bayat E⁵ ; Malekshahi ZV¹ ; Nematollahi L⁵ ; Negahdari B¹
Source: Biointerface Research in Applied Chemistry Published:2023

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. © 2023 by the authors. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).