Trimming glycans as a strategy to produce high levels of functional RBD antigen

IDROVO-HIDALGO, TOMMY; PIGNATARO, MARÍA F.; BREDESTON, LM; ELIAS, FERNANDA; HERRERA, MARÍA G.; PAVAN, MARÍA F.; FOSCALDI, SABRINA; SUIRESZCZ, M; FERNÁNDEZ, NATALIA B.; WETZLER, DIANA E.; PAVAN, C; CRAIG, PATRICIO O.; ROMAN, ERNESTO; RUBERTO, LUCAS A. M.; NOSEDA, DIEGO G.; IBAÑEZ, LORENA I.; CZIBENER, C.; UGALDE, JE; ARGENTINIAN ANTICOVID CONSORTIUM; NADRA, AD; SANTOS, JAVIER; D'ALESSIO, C

Congreso; Pichia 2024; 2024

We have previously reported the expression of the Receptor BindingDomain (RBD) of the SARS-CoV-2 Spike protein in both Picha pastoris andhuman cell systems. P. pastoris is a scalable yeast cost-effective for theproduction of recombinant proteins, which offers the advantages of an efficientsecretion system and assistance in protein folding through the eukaryoticcell's secretory pathway. Although there were differences in size andglycosylation patterns, the structural and conformational characteristics ofthe proteins were highly similar in both systems. However, we found potentialissues related to yeast-added high mannose glycan moieties, as they couldtrigger non-specific immune responses and introduce a high degree of sizeheterogeneity in the protein. We deglycosylated RBD preserving nativeconditions, resulting in a highly pure, uniform, and well-folded monomericprotein with circular dichroism spectra matching those of glycosylated RBD andRBD produced in human cells. The deglycosylated RBD was obtained in a one-stepprocess, yielding high quantities, and was useful to differentiate between serafrom SARS-CoV-2-negative and -positive patients. Furthermore, when used as animmunogen, the deglycosylated variant elicited a humoral immune responsestronger than the glycosylated RBD, produced antibodies with greater neutralizingcapacity, and induced a better cellular response. Trimming glycans afterexpression can be employed to cost-effectively produce numerous proteins thatrequire glycosylation for proper folding and expression, but not forrecognition purposes or activity.