Targeted optimization of single-chain variable fragment (scFv) expression in E. coli using a design-of-experiment approach

Infections by Shiga toxin-producing Escherichia coli (STEC) pose a serious global health risk, potentially leading to hemolytic uremic syndrome (HUS), a life-threatening condition. This study aimed to establish an efficient expression system to produce an anti-Stx2 single-chain variable fragment (scFv) using three E. coli strains: BL21 (DE3), BL21(DE3) pLysS, and ArcticExpress (DE3). Initial tests showed that BL21(DE3) pLysS and ArcticExpress (DE3) produced 0.3–0.4 mg scFv/L using defined media and induction with 0.1 mM IPTG or 10 g/L lactose. A Plackett-Burman design was then used to optimize the concentrations of 2xYT medium, IPTG, and lactose during induction. Under these conditions, ArcticExpress (DE3) reached 19 mg/L of scFv when induced with 1.0 mM IPTG and 2.5 g/L 2xYT. In contrast, BL21(DE3) pLysS yielded 34 mg/L with 1.0 mM IPTG, 0.4 g/L 2xYT, and 0.2 g/L lactose, while IPTG alone resulted in 26 mg/L. The protein yield (Yp/x) also increased from 11.9 to 15.5 mg scFv/g cell. Thus, E. coli BL21(DE3) pLysS showed the highest potential for producing anti-Stx2 scFv, particularly under optimized induction conditions involving IPTG, 2xYT, and lactose. These findings highlight the potential of recombinant E. coli BL21(DE3) pLysS as a robust platform for the scalable production of functional anti-Stx2 scFv fragments. By enabling higher yields under optimized induction strategies, this approach provides an important basis for the development of low-cost diagnostic tools targeting STEC infections, which remain a critical global health challenge.