Navegando por Autor "Piazza, Roxane Maria Fontes"

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    Cost-effective production process of scFv antibody fragments against Shiga toxin 2 via recombinant E. coli
    (Elsevier) Guimarães, Marcela; Luz, Daniela; Augusto, Elisabeth de Fátima Pires; Vieira, Lucia; Costa, Maricilia Silva; Piazza, Roxane Maria Fontes; Pradella, José Geraldo da Cruz
    Shiga toxin (Stx)-producing Escherichia coli (STEC) and its subgroup enterohemorrhagic E. coli are significant pathogens responsible for diarrhea, which can progress to hemorrhagic colitis and hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children. Early diagnosis is crucial for effective clinical man- agement, as antibiotic treatment is not recommended for STEC infections. The present study aimed to establish a cost-effective biotechnological platform for cultivating recombinant E. coli to produce scFv antibody fragments against Stx2 for diagnostic applications. The method was first evaluated through shake flask experiments and subsequently scaled up to bench-scale bioreactors operated in both batch and fed-batch modes using defined culture media. Optimal production conditions were achieved by inducing recombinant E. coli pLys at 18 ◦C for 18 h with 0.1 mM IPTG, resulting in a yield of 3.0 to 4.0 mg scFv/g cell biomass. A fed-batch, high-cell-density procedure with E. coli pLysS achieved a maximum production up to 150 mg scFv/L. A preliminary economic assessment demonstrated the production potential at a value of around $250/g scFv. Economic analysis also highlights that the relative cost of capital investment becomes important as production processes intensify. Therefore, technical parameters such as productivity (scFv mass/bioreactor volume * time) and scFv concentration (mass scFv mass/bioreactor volume) should be prioritized to maximize their values. Similarly, optimization of the recombinant E. coli microbial platform should be pursued to increase the Yp/x level.
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    Targeted optimization of single-chain variable fragment (scFv) expression in E. coli using a design-of-experiment approach
    (Elsevier) Guimaraes, Marcela; Carvalho, Rafaela Vieira; Luz, Daniela; Guilherme, Ariela Pedro Bom; Campos, Claudia Barbosa Ladeira de; Piazza, Roxane Maria Fontes; Pradella, José Geraldo da Cruz
    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.