Improvement of the heterologous production of SARS-CoV2 spike receptor binding domain (RBD) by Pichia pastoris using sorbitol as a co-substrate during DO-Stat induction

SAGUES, J; IDROVO-HIDALGO, T; NOSEDA, DIEGO G.; D'ALESSIO, C; SANTOS, JAVIER; NADRA, ALEJANDRO D.; RUBERTO, LUCAS A. M.

Congreso; Pichia 2024; 2024

We previously reported the production of recombinantSARS-CoV-2 receptor binding domain (RBD) in P. pastoris (yRBD) with similarfeatures as an antigen of that produced in HEK-293T cells. yRBD production in abioreactor reached 520 mg RBD/L. However, pure protein yield was 180 mg/L. Tounderstand the causes of the protein loose the culture supernatant wasincubated in the presence and absence of a protease inhibitor. Without inhibitorthe amount of yRBD was substantially reduced, probably in part due to a lowerpresence of the protein Hisx6 tag. Here, we present an improved procedure toobtain higher amounts of yRBD in a 7L STBR using sorbitol as an induction co-substrate.Fermentations were done using a Low Salt Medium with the following sequence:batch (glycerol 40 g/L), fed-batch (glycerol 600 g/L, DO-Stat mode), transition(2 g/L methanol pulse). During the induction (120 h), either methanol-only fedor methanol/sorbitol co-fed was added, applying a DO-Stat mode in both cases.The co-feeding yielded higher biomass (115 vs 93 g DCW/L), more total proteins(2810 vs 1710 mg/L), and a higher RBD concentration (1434 vs 779 mg/L),increasing the percentage of RBD respect total proteins (51 vs 46%). Volumetricproductivity also improved (9.8 vs 5.3 mg RBD/Lh). The RBD/biomass yield(YRBD/X) was higher (12.4 vs 8.4 mg RBD/g DCW). Additionally, a reduction ofproteolytic activity in the culture supernatant was observed, improving yRBDproduction near 3 times compared to our initial report.