HUMAN RECOMBINANT GALECTIN-1 PRODUCED BY TRANSPLASTOMICS IN TOBACCO PLANTS TO PRODUCE RECOMBINANT HUMAN PROTEIN GALECTIN 1

VATER, CATALINA F ; STUPIRSKI, JUAN C ; BRAVO-ALMONACID, FERNANDO F.; RABINOVICH GABRIEL A; PEREZ SAEZ JUAN M; MORGENFELD M

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Congreso; REUNION ANUAL DE LA SOCIEDAD ARGENTINA DE BIOQUIMICA Y BIOLOGIA MOLECULAR; 2019

SOCIEDAD ARGENTINA DE BIOQUIMICA Y BIOLOGIA MOLECULAR

HUMAN RECOMBINANT GALECTIN-1 PRODUCED BY TRANSPLASTOMICS IN TOBACCO PLANTS TO PRODUCE RECOMBINANT HUMAN PROTEIN GALECTIN 1Vater, Catalina F1.; Stupirski, Juan C2.; Bravo-Almonacid, Fernando F.1; Rabinovich, Gabriel A.2; Pérez Sáez, Juan M.2; Morgenfeld, Mauro M1.1 Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina2Laboratorio de Inmunopatología y Oncoinmunología, Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, C1428ADN Buenos Aires, Argentinacata.vater@gmail.comJ.M.P.S. and M.M.M. contributed equally to this workTransplastomics plants stand out from other molecular farming platforms because of their potentiallythe highly efficient production of recombinant proteins (>50% of the total soluble protein). In some cases, however, low and even undetectable levels of heterologous protein expression have been reported in this system. This fact makes it necessary to evaluate and study the expression system whenever a new protein emerges as a candidate to be produced using this platform. The aim of this work, therefore, was to assess the feasibility of producing transplastomic plants that express a human protein with biological immunomodulatory activity and potential therapeutic use. For this purpose we chose to expressed human Galectin 1 (hGal-1), a carbohydrate- binding protein with proven immunomodulatory and anti-inflammatory activities. This protein has great broad therapeutic potential and has the appropriate biochemical characteristics to be expressed in the plastid system.Initially, we cloned the LGALS1 gene in a plastid transformation vector (pBSW5?UTRGal1). This vector was used to transform Nicotiana tabacum leaves using biolistics. Transplastomic lines, obtained from three independent recombination events (verified by PCR), were characterized at the molecular level. In order to confirm the homoplasty of the lines we performed a Southern blot assay. We corroborated the transgene transcriptional activity by Northern blot. The expression of hGal-1 in the transplastomic plants was analyzed by Western blot and quantified by ELISA. In both cases we used a specific polyclonal antibody anti-Gal1 polyclonal antibody for detection and recombinant hGal-1 expressed in E. coli as positive control. Standard protocol of protein extraction was adapted in order to increase the recovery of active Gal-1 by adding a reducing agent. This recombinant protein, accumulated in the soluble protein fraction of the transplastomic plants, was finally successfully purified by an affinity chromatography with a lactosyl-Ssepharose column.In conclusion, homoplastic tobacco plants capable of producing hGal-1 were obtained. Human Galectin 1 produced by tobacco chloroplasts was electrophoretically indistinguishable from bacterial hGal-1. Purification by affinity chromatography proves demonstrated intactthat the carbohydrate recognition domain is intact, indicating suggesting that preservation of the biochemical activity of the recombinant hGal-1 is preserved. Further in vitro and in vivo experiments are being currently in processperformed in order to corroborate the biological activity of the recombinant protein. Given the therapeutic potential of this protein in the treatment of autoimmune and chronic inflammatory disorders, this new expression system may serve to produce endotoxin-free, hGal-1 for pre-clinical and clinical studies.