Recombinant Protein Production in CHOZN Cells

CHOZN GS−/− cells (Merck) were maintained in suspension cultures in serum-free media (EX-CELL CHO CD Fusion, Merck) supplemented with 4 mM L-glutamine, as previously described (Tian et al., 2019 (link)). An expression construct containing the entire coding sequence of human AGA was synthesized by Genewiz, United States. Full-length cDNA of human GUSB, TPP1, and GAA were purchased from Horizon Discovery, United Kingdom, while human IDS cDNA was purchased from Sino Biological. C-terminal His-tagged CTSD was produced as previously reported (Marques et al., 2020 (link)). All reporter constructs were cloned into pCGS3 (Merck). Cells were seeded at a density of 0.5 × 10⁶ cells/mL in T25 flasks (NUNC) 24 h prior to transfection. Approximately 2 × 10⁶ cells were transfected with 8 μg endotoxin-free plasmids using Amaxa kit V and program U24 with Amaxa Nucleofector 2B (Lonza). 72 h post-transfection, cells were plated at 500–1,000 cells/well in 96-wells in 200 μL Minipool Plating Medium containing 80% EX-CELL^® CHO Cloning Medium (Merck) and 20% EX-CELL CD CHO Fusion media without glutamine for selection. Screening of high expression minipools were performed by determining enzyme activity in spent media for AGA, GUSB, GAA and IDS, by SDS-PAGE for TPP1 and CTSD. Selected minipools were further single cell sorted by fluorescence-activated cell sorting (FACS) (Sony) and expanded in 50 mL TPP TubeSpin^® shaking Bioreactors (180 rpm, 37°C and 5% CO₂).