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Targeted optimization of single-chain variable fragment (scFv) expression in E. coli using a design-of-experiment
Marcela Guimarães1, Rafaela Vieira Carvalho1, Daniela Luz2
1Instituto de Ciência e Tecnologia, Universidade Federal de São Paulo, Brazil.
International Journal of Biological Macromolecules
|September 27, 2025
Summary
Shiga toxin-producing E. coli (STEC) infections cause severe illness. This study optimized E. coli BL21(DE3) pLysS to efficiently produce anti-Stx2 single-chain variable fragments (scFv) for potential diagnostic tools.
Area of Science:
- Biotechnology
- Molecular Biology
- Microbiology
Background:
- Shiga toxin-producing Escherichia coli (STEC) infections represent a significant global health threat, potentially leading to severe complications like hemolytic uremic syndrome (HUS).
- Developing effective countermeasures, including diagnostic tools, is crucial for managing STEC outbreaks.
- Recombinant antibody fragments, such as single-chain variable fragments (scFv), offer potential for targeted therapeutic and diagnostic applications against bacterial toxins.
Purpose of the Study:
- To establish and optimize an efficient expression system for producing anti-Stx2 single-chain variable fragment (scFv) in Escherichia coli.
- To compare the production yields of anti-Stx2 scFv in different E. coli strains (BL21(DE3), BL21(DE3) pLysS, ArcticExpress (DE3)).
- To identify optimal induction conditions for maximizing scFv production in the most suitable E. coli strain.
Main Methods:
- Expression of anti-Stx2 scFv was evaluated in three E. coli strains: BL21(DE3), BL21(DE3) pLysS, and ArcticExpress (DE3).
- Initial expression levels were assessed under standard induction conditions using IPTG and lactose.
- A Plackett-Burman experimental design was employed to optimize induction parameters, including 2xYT medium concentration, IPTG, and lactose concentrations.
Main Results:
- E. coli BL21(DE3) pLysS and ArcticExpress (DE3) initially produced 0.3-0.4 mg scFv/L.
- Optimization using Plackett-Burman design significantly enhanced yields.
- E. coli BL21(DE3) pLysS achieved the highest yield of 34 mg/L under optimized conditions (1.0 mM IPTG, 0.4 g/L 2xYT, 0.2 g/L lactose), with an increased protein yield per cell (Yp/x) from 11.9 to 15.5 mg scFv/g cell.
Conclusions:
- E. coli BL21(DE3) pLysS demonstrates superior potential as a host for the scalable production of functional anti-Stx2 scFv.
- Optimized induction strategies involving IPTG, 2xYT medium, and lactose are key to achieving high yields.
- This optimized expression system provides a foundation for developing cost-effective diagnostic tools to combat STEC infections.
