Expression of Galectin-8 in mammalian cells by lentiviral transduction

JULIETA CARABELLI ; OSCAR CAMPETELLA; DIEGO ALVAREZ; MARIA VIRGINIA TRIBULATTI; PAULA JIGENA; ALEJANDRO CASSOLA

San Martin

Simposio; 3rd Argentinian Symposium on Glycobiology; 2019

Galectin-8 (Gal-8) is a mammalian lectin capable of stimulatingthe elicitation of antigen-specific immune responses, whichmakes it a good candidate to use as an additive in vaccineformulations. The aim of the present project is the expression andcharacterization of Gal-8 in HEK293 cell line by lentiviraltransduction. Despite galectins lack a signal peptide they areactually secreted as soluble proteins to the extracellular milieu bya non-conventional pathway. Therefore, we designed twodifferent constructs to generate recombinant Gal-8: i) a constructcontaining a signal peptide to obtain a ?secreted? form and, ii) aconstruct without signal peptide for the ?intracellular? form. Sinceit was previously reported that Gal-8 sequence has two putativesites for N-glycosylation, we first analyzed the differences in theelectrophoretic mobility between the ?secreted? (obtained formcell culture supernatants) and ?intracellular? (obtained form cellculture supernatants or cell extracts) forms. After determiningthat the ?secreted? Gal-8 is glycosylated, we evaluated bydifferent functional assays whether glycosylation affected proteinactivity, using the validated recombinant Gal-8 expressed in E.coli as a reference. Lactosylsepharose ?pull down? and cellsurface binding assays demonstrated that both ?secreted? and?intracellular? forms of Gal-8 expressed in HEK293 cells retainedthe expected galectin activity.