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Microorganisms 2018, 6(2), 37; https://doi.org/10.3390/microorganisms6020037

Recombinant Inga Laurina Trypsin Inhibitor (ILTI) Production in Komagataella Phaffii Confirms Its Potential Anti-Biofilm Effect and Reveals an Anti-Tumoral Activity

1
Grupo de Engenharia de Biocatalizadores, Instituto de Ciências Biológicas, Universidade de Brasília, CEP 70.790-900 Brasília-DF, Brazil
2
Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, CEP 70.790-160 Brasília-DF, Brazil
3
Faculdade de Ciências Farmacêuticas, Alimentos e Nutrição-UFMS, Laboratório de Purificação de Proteínas e suas Funções Biológicas-LPPFB, Cidade Universitária S/N-Caixa Postal 549, CEP 79.070-900 Campo Grande-MS, Brazil
4
Instituto de Ciências Exatas e Tecnologia, Universidade Federal do Amazonas, Itacoatiara, CEP 69100-000 Amazonas, Brazil
5
S-Inova Biotech, Universidade Católica Dom Bosco, Programa de Pós-graduação em Biotecnologia, Campo Grande, CEP 79117-900 Mato Grosso do Sul, Brazil
6
Centro de Análises Proteômicas e Bioquímicas. Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, CEP 70.790-160 Brasília, Distrito Federal, Brazil
7
Programa de Pós-graduação em Patologia Molecular, Universidade de Brasília, CEP 70.790-900 Brasília, Distrito Federal, Brazil
*
Author to whom correspondence should be addressed.
Received: 30 March 2018 / Revised: 23 April 2018 / Accepted: 24 April 2018 / Published: 28 April 2018
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Abstract

Protease inhibitors have a broad biotechnological application ranging from medical drugs to anti-microbial agents. The Inga laurina trypsin inhibitor (ILTI) previously showed a great in vitro inhibitory effect under the adherence of Staphylococcus species, being a strong candidate for use as an anti-biofilm agent. Nevertheless, this is found in small quantities in its sources, which impairs its utilization at an industrial scale. Within this context, heterologous production using recombinant microorganisms is one of the best options to scale up the recombinant protein production. Thus, this work aimed at utilizing Komagataella phaffii to produce recombinant ILTI. For this, the vector pPIC9K+ILTI was constructed and inserted into the genome of the yeast K. phaffii, strain GS115. The protein expression was highest after 48 h using methanol 1%. A matrix-assisted laser desorption ionization–time-of-flight (MALDI–TOF) analysis was performed to confirm the production of the recombinant ILTI and its activity was investigated trough inhibitory assays using the synthetic substrate Nα-Benzoyl-D,L-arginine p-nitroanilide hydrochloride (BAPNA). Finally, recombinant ILTI (rILTI) was used in assays, showing that there was no significant difference between native and recombinant ILTI in its inhibitory activity in biofilm formation. Anti-tumor assay against Ehrlich ascites tumor (EAT) cells showed that rILTI has a potential anti-tumoral effect, showing the same effect as Melittin when incubated for 48 h in concentrations above 25 µg/mL. All together the results suggests broad applications for rILTI. View Full-Text
Keywords: Komagataella phaffii; heterologous expression; Inga laurina trypsin inhibitor (ILTI); biofilm assay; anti-tumor effect Komagataella phaffii; heterologous expression; Inga laurina trypsin inhibitor (ILTI); biofilm assay; anti-tumor effect
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Carneiro, F.C.; Weber, S.S.; Silva, O.N.; Jacobowski, A.C.; Ramada, M.H.S.; Macedo, M.L.R.; Franco, O.L.; Parachin, N.S. Recombinant Inga Laurina Trypsin Inhibitor (ILTI) Production in Komagataella Phaffii Confirms Its Potential Anti-Biofilm Effect and Reveals an Anti-Tumoral Activity. Microorganisms 2018, 6, 37.

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