Solubility assessment of single-chain antibody fragment against epithelial cell adhesion molecule extracellular domain in four Escherichia coli strains

BACKGROUND: Overexpression of the EpCAM (epithelial cell adhesion molecule) in malignancies makes it an attractive target for passive immunotherapy in a wide range of carcinomas. In comparison with full-length antibodies, due to the small size, the scFvs (single-chain variable fragments) are more suitable for recombinant expression in E. coli (Escherichia coli). However, the proteins expressed in large amounts in E. coli tend to form inclusion bodies that need to be refolded which may result in poor recovery of bioactive proteins. Various engineered strains were shown to be able to alleviate the insolubility problem. Here, we studied the impact of four E. coli strains on the soluble level of anti-EpEX-scFv (anti-EpCAM extracellular domain-scFv) protein. RESULTS: Although results showed that the amount of soluble anti-EpEX-scFv obtained in BL21(TM) (DE3) (114.22 ± 3.47 mg/L) was significantly higher to those produced in the same condition in E. coli Rosetta(TM) (DE3) (71.39 ± 0.31 mg/L), and Origami(TM) T7 (58.99 ± 0.44 mg/L) strains, it was not significantly different from that produced by E. coli SHuffle(TM) T7 (108.87 ± 2.71 mg/L). Furthermore, the highest volumetric productivity of protein reached 318.29 ± 26.38 mg/L in BL21(TM) (DE3). CONCLUSIONS: Although BL21(TM) (DE3) can be a suitable strain for high-level production of anti-EpEX-scFv protein, due to higher solubility yield (about 55%), E. coli SHuffle(TM) T7 seems to be better candidate for soluble production of scfv compared to BL21(TM) (DE3) (solubility yield of about 30%).