Recombinant expression and purification of TANB77-derived pilin-like proteins 935 and 938

Increasing evidence highlights the gut microbiota as a source of diagnostic biomarkers and a key modulator of anti-tumor immunity and cancer immunotherapy responses. Pilin-like proteins from the uncultured TANB77 clade have shown potential to enhance immune checkpoint blockade therapy, yet protocols for their recombinant production have not been established. Here, we present the first optimized method for the expression and purification of two TANB77-derived pilin-like proteins, 935 and 938, using the Escherichia coli (E. coli) BL21(DE3)/pET system. The workflow integrates optimized expression conditions, two-step purification by Ni-NTA affinity and size-exclusion chromatography (SEC), and endotoxin removal for downstream applications. We reveal significant differences in the expression profiles and biophysical properties of the two proteins: protein 935 was expressed at high levels (∼2.5 mg/100 mL) and formed stable oligomers. In contrast, protein 938 exhibited >30-fold lower yields (∼0.08 mg/100 mL) and significant fragmentation, indicating intrinsic instability within this expression system. This study provides the first systematic framework for producing TANB77 pilin-like proteins, facilitating future exploration of their biochemical properties and therapeutic potential in cancer immunotherapy.