Heterologous expression of Homo sapiens alpha-folate receptors in E. coli by fusion with a trigger factor for enhanced solubilization

Autor(es)

Messias Miranda, Beatriz Nogueira ^{[1, 2]} ; Fotoran, Wesley Luzetti ^[3] ; Canduri, Fernanda ^[1] ; Ferreira Souza, Dulce Helena ^[4] ; Wunderlich, Gerhard ^[3] ; Carrilho, Emanuel ^{[1, 2]}

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Inst Quim Sao Carlos, Av Trabalhador Seo Carlense 400, BR-13566590 Sao Carlos, SP - Brazil

^[2] Inst Nacl Ciencia & Tecnol Bioanalit INCTBio, BR-13083970 Campinas, SP - Brazil

^[3] Univ Sao Paulo, Inst Ciencias Biomed, Av Prof Lineu Prestes, 1374 Cidade Univ, Sao Paulo, SP - Brazil

^[4] Univ Fed Sao Carlos, Dept Quim, Rodovia Washington Luis S-N, Sao Carlos, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Protein Expression and Purification; v. 142, p. 75-80, FEB 2018.
• Citações Web of Science: 1

Resumo

The role of Alpha folate receptors (FR alpha) in folate metabolism and cancer development has been extensively studied. The reason for this is not only associated to its direct relation to disease development but also to its potential use as a highly sensitive and specific biomarker for cancers therapies. Over the recent years, the crystal structures of human FR alpha complexed with different ligands were described relying on an expensive and time-consuming production process. Here, we constructed an efficient system for the expression and purification of a human FR alpha in E. coli. Unlike a conventional expression method we used a specific protein fusion expressing the target protein together with a trigger factor (TF). This factor is a chaperone from E. coli that assists the correct folding of newly synthesized polypeptide chains. The activity of rTFFR alpha was comparable to glycosylphosphatidylinositol (GPI) anchored proteins extracted from HeLa tumor cells. Our work demonstrates a straightforward and versatile approach for the production of active human FR alpha by heterologous expression; this approach further enhances the development of inhibition studies and biotechnological applications. The purified product was then conjugated to liposomes, obtaining a 35% higher signal from densitometry measurement on the immunoblotting assay in the contruct containing the Ni-NTA tag, as a mimesis of an exosome, which is of vital importance to nanotherapeutic techniques associated to treatment and diagnosis of tumors. (AU)

• Processo FAPESP: 11/16364-6 - Clonagem, expressão e purificação de alfa receptores de folato de Homo sapiens para aplicações bioanalíticas em câncer
• Beneficiário: Beatriz Nogueira Messias de Miranda
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 08/57805-2 - Instituto de Bioanalítica
• Beneficiário: Lauro Tatsuo Kubota
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 13/15892-4 - Produção de vesículas poliméricas para aplicações bioanalíticas em câncer
• Beneficiário: Beatriz Nogueira Messias de Miranda
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Mestrado
• Processo FAPESP: 09/54040-8 - EMU: aquisição de um espectrômetro de massas de alta resolução Orbitrap para descoberta e elucidação estrutural compostos biologicamente ativos: aplicações em proteômica e biomarcadores, síntese, isolamento e caracterização de produtos naturais, estudos de sistemas redox em alimentos e síntese enzimática
• Beneficiário: Emanuel Carrilho
• Modalidade de apoio: Auxílio à Pesquisa - Programa Equipamentos Multiusuários