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

Protease inhibitors have a broad biotechnological application ranging from medical drugs to anti-microbial agents. The trypsin inhibitor (ILTI) previously showed a great in vitro inhibitory effect under the adherence of species, being a strong candidate for use as an anti-biofilm agent. Nevertheless...

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Bibliographic Details
Published in:	Microorganisms (Basel) Vol. 6; no. 2; p. 37
Main Authors:	Carneiro, Fábio C, Weber, Simone S, Silva, Osmar N, Jacobowski, Ana Cristina, Ramada, Marcelo H S, Macedo, Maria L R, Franco, Octávio L, Parachin, Nádia S
Format:	Journal Article
Language:	English
Published:	Switzerland MDPI AG 28-04-2018 MDPI
Subjects:	Anticancer properties Antiinfectives and antibacterials Arginine Ascites Assaying Biofilms Biotechnology Ehrlich ascites tumor Gene expression Genomes Ionization Microorganisms Protease inhibitors Proteinase inhibitors Proteins Seeds Substrate inhibition Trypsin Trypsin inhibitors Tumors Yeast Yeasts Brazil United States > US Komagataella phaffii heterologous expression biofilm assay anti-tumor effect Inga laurina trypsin inhibitor (ILTI)
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Summary:	Protease inhibitors have a broad biotechnological application ranging from medical drugs to anti-microbial agents. The trypsin inhibitor (ILTI) previously showed a great in vitro inhibitory effect under the adherence of 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 to produce recombinant ILTI. For this, the vector pPIC9K+ILTI was constructed and inserted into the genome of the yeast , 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	2076-2607 2076-2607
DOI:	10.3390/microorganisms6020037