OPTIMIZATION OF LEISHMANIA TARENTOLAE FOR PRODUCTION OF THERAPEUTIC GLYCOPROTEIN DRIVEN BY GENOMICS, PROTEOMICS AND METABOLOMICS INTEGRATION AND EFFECTS OF GENE COPY NUMBER

Renato Lima Senra; Luana Maria Pacheco Schittino; Andrea de Oliveira Barros Ribon; JACQUELINE ARAUJO FIUZA; Tiago Antônio de Oliveira Mendes

Paper Title

Authors

Renato Lima Senra
Luana Maria Pacheco Schittino
Andrea de Oliveira Barros Ribon
JACQUELINE ARAUJO FIUZA
Tiago Antônio de Oliveira Mendes

Modality

Poster

Subject area

Systems Biology and Modeling

Publishing Date

21/11/2024

Country of Publishing

Brazil | Brasil

Language of Publishing

Inglês

Paper Page

https://www.even3.com.br/anais/xmeeting-2024/831591-optimization-of-leishmania-tarentolae-for-production-of-therapeutic-glycoprotein-driven-by-genomics-proteomics-a

ISBN

978-65-272-0843-3

Keywords

Leishmania tarentolae, glycoproteins, heterologous expression, sialylation

Summary

The production of therapeutic glycoproteins is expensive due to need to use mammalian cell culture that depend of complex and expensive culture media and have low yield of production. Leishmania tarentolae, a non-pathogenic trypanosomal protozoan for mammals, has been suggested as an alternative system for heterologous expression of glycoproteins due to the existence of efficient methods for heterologous expression, with low cost. The glycosylation profile of proteins expressed in L. tarentolae is similar to mammals, there are some differences in the carbohydrate profile present in the endings of the glycidic chains due to the absence of biosynthesis enzymes and the incorporation of sialic acid. The present project proposed the development of an expression platform of interferon-ß (IFN- ß) using genetically optimized L. tarentolae strains for glycoprotein synthesis with potential sialic acid incorporation at the end of the glycine chain providing a more effective action of proteins with reduced costs. Approaches of genomic and metabolomics were used to understand more about the metabolic pathways found in these organisms that indicated an alternative to make the L. tarentolae expression system more optimized. Protein sequences involved in the metabolic reactions of N-glycan biosynthesis were retrieved from KEGG. With Blastp was compared with the H. sapiens proteins against the predicted L. tarentolae proteome from the TritrypDB database. The sequences of the genes of interest were designed with optimized códons. Also, to investigate the impact of gene copy number variation on transcripts and protein expression, was developed constructions of plasmids with a three varied selection of expression markers and generation of strains containing different numbers of these vectors. The genetically modified strains were obtained by transfection with genes optimized for the genus Leishmania and confirmed by PCR, qPCR, SDS-PAGE and Western Blot labeling with specific antibodies (anti-his) confirmed heterologous protein production and purification by affinity chromatography in the selected lineages. Through the analysis of mass spectrometry, it was possible to recognize the glycosylation signal and differentiate the lineages. In vitro IFN-ß activity assays showed the efficiency of the protein produced in the optimized system to reduce the inflammatory effect in relation to other molecules produced in other expression systems. A correlation was established between the number of copies of the transfected gene and the resulting transcript and protein levels in L. tarentolae and were suggested possibilities of other metabolic and enzymatic targets for optimizing protein expression in this organism Thus, in this work was developed a more accessible expression platform of glycoproteins with biochemical and pharmacokinetic properties more similar to the organism of origin using L. tarentolae.

Title of the Event: 20º Congresso Brasileiro de Bioinformática: X-Meeting 2024
City of the Event: Salvador
Title of the Proceedings of the event: X-Meeting presentations
Name of the Publisher: Even3
Means of Dissemination: Meio Digital

How to cite

SENRA, Renato Lima et al.. OPTIMIZATION OF LEISHMANIA TARENTOLAE FOR PRODUCTION OF THERAPEUTIC GLYCOPROTEIN DRIVEN BY GENOMICS, PROTEOMICS AND METABOLOMICS INTEGRATION AND EFFECTS OF GENE COPY NUMBER.. In: X-Meeting presentations. Anais...Salvador(BA) Hotel Deville Prime, 2024. Available in: https//www.even3.com.br/anais/xmeeting-2024/831591-OPTIMIZATION-OF-LEISHMANIA-TARENTOLAE-FOR-PRODUCTION-OF-THERAPEUTIC-GLYCOPROTEIN-DRIVEN-BY-GENOMICS-PROTEOMICS-A. Access in: 07/05/2025