HUMAN RECOMBINANT GALECTIN 1 PRODUCED BY TRANSPLASTOMIC TOBACCO PLANTS

BRAVO-ALMONACID FERNANDO F; MORGENFELD MAURO MIGUEL; STUPRIRSKI JUAN CARLOS; PÉREZ SAÉZ JUAN MANUEL; VATER CATALINA FRANCISCA; RABINOVICH GABRIEL A

Salta

Congreso; Reunión Conjunta por el XIV Congreso de la Pan-American Association for Biochemistry and Molecular Biology y LV Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2019

Sociedad Argentina de Investigaciones en Bioquimica y Biología. Pan-American Association for Biochemistry and Molecular Biology

HUMAN RECOMBINANT GALECTIN 1 PRODUCED BY TRANSPLASTOMIC TOBACCO PLANTSVater CF1, Stupirski JC2, Bravo-Almonacid FF1, Rabinovich GA2, Pérez Sáez JM2*, Morgenfeld MM1**These authors contributed equally to this work.1Laboratorio de Biotecnología Vegetal (INGEBI-CONICET), 2 Laboratorio de Inmunopatología y Oncoinmunología (IBYME-CONICET)E-mail: mmorgen@dna.uba.arTransplastomic plants stand out from other molecular farming platforms because of the 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 immunomodulatory activity and potential therapeutic use. For this purpose, we expressed human Galectin 1 (hGal-1), a carbohydrate-binding protein with proven immunomodulatory and anti-inflammatory activities. This protein has broad therapeutic potential and the appropriate biochemical characteristics to be expressed in the plastid system. Initially, we cloned the LGALS1 gene in a plastid transformation vector (pBSW5?UTR). 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. We corroborated the transgene transcriptional activity by Northern blot. The expression of hGal-1 in transplastomic plants was analyzed by Western blot and quantified by ELISA. In both cases, we used a specific anti-Gal1 polyclonal antibody for detection and recombinant hGal-1 expressed in Escherichia coli as the positive control. The 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 purified by affinity chromatography with a lactosyl-Sepharose 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 demonstrated an intact carbohydrate recognition domain, suggesting preservation of the biochemical activity of the recombinant hGal-1. Further in vitro and in vivo experiments are currently in process 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.