Fig. 4

Nanobody production with protease inhibition and functional evaluation. T. reesei E1AB1-XA3 strain based transformants were cultivated in shake flasks on a non-inducing medium containing 3% glucose and with (+)/without (−) protease inhibitor cocktail. a Western blotting analysis using anti-His tag antibody on gels that were performed on SDS-PAGE loaded with 5 µL of 8-fold diluted supernatant. Closed triangles indicate the target nanobody as estimated from the molecular weight. Open triangles indicate the un-cleaved protein at the KEX2 linker, corresponding to the CBD-1ZVH fusion protein. Dashed triangles indicate cleaved nanobodies. b Concentration of nanobodies secreted to culture supernatant at day 4. The nanobodies concentration were determined by western blotting using the purified nanobody as a calibration curve. c Purified His-tagged nanobodies using Ni-NTA beads. Protease inhibitor-added culture supernatant from day 4 were used for purification. d Kinetics analysis of 1ZVH/lysozyme and caplacizumab/vWF. Association and dissociation rate were measured by reacting the sensor with nanobody conjugated via His-tag and various concentrations of antigen. Error bars indicate standard deviations. Statistical significance was determined by a two-tailed unpaired Student’s t-test. *p < 0.05. **p < 0.01