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

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dc.contributor.authorGuimaraes, Marcela
dc.contributor.authorCarvalho, Rafaela Vieira
dc.contributor.authorLuz, Daniela
dc.contributor.authorGuilherme, Ariela Pedro Bom
dc.contributor.authorCampos, Claudia Barbosa Ladeira de
dc.contributor.authorPiazza, Roxane Maria Fontes
dc.contributor.authorPradella, José Geraldo da Cruz
dc.date.accessioned2026-02-12T18:36:28Z
dc.date.available2026-02-12T18:36:28Z
dc.date.issued22025
dc.description.abstractInfections 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.
dc.description.physical13 p.
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.description.uriFAPESP: 2019/25623-7, 2021/12282-7
dc.format.mimetypePDF
dc.identifier.affiliationUniversidade do Vale do Paraíba
dc.identifier.affiliationInstituto Butantan
dc.identifier.affiliationUniversidade Federal de São Paulo
dc.identifier.bibliographicCitationGUIMARÃES, M. et al. Targeted optimization of single-chain variable fragment (scFv) expression in E. coli using a design-of-experiment approach. International Journal of Biological Macromolecules, v. 329, p. 1-13, 2025. Disponível em: 10.1016/j.ijbiomac.2025.147881.
dc.identifier.doi10.1016/j.ijbiomac.2025.147881
dc.identifier.urihttps://repositorio.univap.br/handle/123456789/1149
dc.language.isoen_US
dc.publisherElsevier
dc.rights.holderInternational Journal of Biological Macromolecules
dc.subject.keywordscFv anti-Stx2
dc.subject.keywordE. coli
dc.subject.keywordInduction optimization
dc.titleTargeted optimization of single-chain variable fragment (scFv) expression in E. coli using a design-of-experiment approach
dc.typeArtigos de Periódicos

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