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Heterologous expression of therapeutic protein in filamentous fungi

Paula Macedo Cunha Rolf Alexander Prade; Fernando Segato

Anais do 32º Congresso Brasileiro de Microbiologia 2023 n.990-1, 2023

Foz do Iguaçú-PR SBM 2023

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  • Título:
    Heterologous expression of therapeutic protein in filamentous fungi
  • Autor: Paula Macedo Cunha
  • Rolf Alexander Prade; Fernando Segato
  • Assuntos: MICROBIOLOGIA; BIOTECNOLOGIA; INDÚSTRIA FARMACÊUTICA; Heterologous Expression; Pharmaceutical Industry; Timps
  • É parte de: Anais do 32º Congresso Brasileiro de Microbiologia 2023 n.990-1, 2023
  • Notas: Disponível em: https://sbmicrobiologia.org.br/32cbm-anais/resumos/R-0990-1.html. Acesso em: 30 nov. 2023.
  • Descrição: Recombinant proteins, widely used in food and biofuel industries, are increasingly gaining importance in the pharmaceutical industry due to their versatility and specificity in treating various diseases. Several cell types have been utilized for expressing and producing human recombinant proteins, including Escherichia coli, Saccharomyces cerevisiae, and mammalian cells. This study aims to produce proteins from the Tissue Inhibitor of Metalloproteases (TIMP) family using the filamentous fungus Aspergillus nidulans. TIMPs are natural inhibitors of Matrix Metalloproteinases (MMPs), and their imbalance in the human body leads to several pathological processes involving connective tissue degradation. We amplified synthetic optimized genes TIMP1 (T1) and TIMP3 (T3) using PCR, fused them with proteins from the Glycoside Hydrolase family (GH7.1 and GH5.1), and utilized a TEV protease as a linker. The constructions were inserted into the pEXPYR vector using Gibson Assembly method and cloned into E. coli via heat shock technique. Subsequently, positive plasmids were introduced into the A. nidulans through PEG-mediated transformation. Positive transformants were confirmed using a reverse zymogram assay, and their activity was assessed by FRET assay against MMP2. Transformants were cultivated in minimal media added by 2, 3, and 5% maltose as inducer with and without agitation for up to 6 days. The results yielded three positive transformants: fGH7T1, fGH7T3, and fGH5T3. Upon inducing protein production in maltose, fGH7T1 showed better production in 2 days under all tested conditions without agitation, while with agitation, it was only produced at 3% on the third day. fGH7T3 exhibited higher protein production in 2 and 3% maltose with agitation for 6 days, but it also produced well when grown for 2 days with 2 and 3% maltose without agitation. This form of production also showed fGH7T3 cleavage from the thir
  • Editor: Foz do Iguaçú-PR SBM
  • Data de criação/publicação: 2023
  • Formato: n. 990-1.
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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