Galectin expression in mammalian cell system

BELEN ESTRUCH; CECILIA ARAHI PRATO; MARÍA VIRGINIA TRIBULATTI

Santa Fe

Simposio; IX SLATCC 2022; 2022

Universidad Nacional de Litoral

Galectins (Gals), a family of mammalian lectins, have become key soluble players in theimmune system. Despite Gals do not possess a signal peptide, they are readily secreted intothe extracellular space by an unconventional mechanism. Galectin-8 (Gal-8), in particular,displays multiple immunostimulatory properties which makes it an interesting candidate to beused as an adjuvant in vaccine formulations.The present work aims to express recombinant Gal-8 in an endotoxin-free system and test itsactivity in the immune response. For this purpose, we expressed recombinant Gal-8 in theHEK293 cell line by lentiviral transduction with the addition of a heterologous signal peptide(Sec-pLB-Gal-8) to enhance protein secretion and optimize the purification step from theculture supernatant. Despite Gals are not glycosylated, they present potential glycosylationsites in their sequences. Therefore, we also included in our study the expression ofrecombinant Gal-8 without signal peptide (pLB-Gal-8) to compare protein function with thesignal peptide-containing version.As a result, we obtained a high efficiency (> 80%) stable-transduced HEK293 cell line thatsecretes Gal-8 to the extracellular milieu. The galectin bound to Lactosyl-Sepharose resin andwas eluted with lactose, indicating that lectin-activity is preserved. Moreover, secreted Gal-8possessed an apparent molecular weight higher than the intracellular version without thesignal peptide, compatible with glycosylation. Cell culture conditions were optimized toaugment recombinant Gal-8 expression and, even at a low-scale lab production, the proteinyield was consistent to perform molecular and functional characterization.