Enhanced production of secretory glycoprotein VSTM1-v2 with mouse IgGκ signal peptide in optimized HEK293F transient transfection

• Mouse IgGκ signal peptide greatly enhanced secretion capacity of VSTM1-v2.
• The yield of VSTM1-v2 in the optimized suspension HEK293F transient expression system was increased by 7 folds.
• VSTM1-v2 was synthesized and secreted as a hyper-glycosylated protein with complex oligosaccharides.
• The purified VSTM1-v2 exhibited excellent bioactivity.
• The study provides a high-efficient way to rapidly enhance production of proteins with weak secretion capacity.

VSTM1-v2 is a secretory glycoprotein identified by our laboratory. Our previous study revealed that VSTM1-v2 could promote differentiation and activation of Th17 cells. To explore the role of VSTM1-v2 in the immune system further, a source of abundant high-quality recombinant protein is warranted. However, high-level expression of bioactive VSTM1-v2 is difficult due to its weak secretion capacity. To obtain sufficient recombinant VSTM1-v2, we developed an improved expression and purification system by replacing the native signal peptide with a mouse IgGκ signal peptide that did not alter the protein cleavage site. We also optimized parameters for a transient gene expression system in HEK293F cells suspended in serum-free media with polyethyleneimine. Finally, 3.6 mg/L recombinant VSTM1-v2 protein with N-glycosylation and no less than 95% purity was obtained through one-step purification with Ni affinity chromatography. The final yield after purification was increased by more than 7-fold compared to the yield from our previously reported HEK293T system (from 0.5 mg/L to 3.6 mg/L). More importantly, VSTM1-v2 protein exhibited excellent bioactivity. In conclusion, the improved system is not only a dependable source of abundant bioactive VSTM1-v2 for functional studies but also demonstrates a highly efficient approach for enhancing the production of proteins in a short time period, especially for secretory proteins with poor yields.