Recombinant PilS: Cloning, Expression and Biochemical Characterization of a Pil-Fimbriae Subunit

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Munhoz, Danielle D. ; Silva, Jessika C. A. ; Freitas, Natalia C. ; Iwai, Leo K. ; Aires, Karina A. ; Ozaki, Christiane Y. ; Souza, Cristiane S. ; Rocha, Leticia B. ; Silva, Miriam A. ; Henrique, Izabella M. ; Elias, Waldir P. ; Carvalho, Eneas ; Morganti, Ligia ; Chura-Chambi, Rosa M. ; Piazza, Roxane M. F.

• Document type: Journal article
• Source: MICROORGANISMS; v. 10, n. 6, p. 17-pg., 2022-06-01.

Abstract

Pil-fimbriae is a type IV pili member, which is a remarkably versatile component with a wide variety of functions, including motility, attachment to different surfaces, electrical conductance, DNA acquisition, and secretion of a broad range of structurally distinct protein substrates. Despite the previous functional characterization of Pil, more studies are required to understand the regulation of Pil expression and production, since the exact mechanisms involved in these steps are still unknown. Therefore it is extremely important to have a protein with the correct secondary and tertiary structure that will enable an accurate characterization and a specific antisera generation. For this reason, the aim of this work was to generate potential tools for further investigations to comprehend the mechanisms involved in Pil regulation and its role in pathogenic E. coli infections with the obtaining of a precise native-like recombinant PilS and the corresponding antisera. The pilS gene was successfully cloned into an expression vector, and recombinant PilS (rPilS) was efficiently solubilized and purified by metal affinity chromatography. Protein characterization analyses indicated that rPilS presented native-like secondary and tertiary structures after the refolding process. The generated anti-rPilS sera efficiently recognized recombinant and native proteins from atypical enteropathogenic E. coli strains. (AU)

• FAPESP's process: 09/14845-7 - Immunodiagnosis of diarrheagenic Escherichia coli
• Grantee: Roxane Maria Fontes Piazza
• Support Opportunities: Regular Research Grants
• FAPESP's process: 04/12136-5 - Atypical enteropathogenetic Escherichia coli (atypical EPEC)
• Grantee: Waldir Pereira Elias Junior
• Support Opportunities: Research Projects - Thematic Grants