21/08/1402
Optimization of the Expression of Recombinant Cetuximab Single-Chain Fragment Variable and Comparative its Purification with Magnetic Nanoparticles and Conventional Fast Protein Liquid Chromatography
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.