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