Fig. 5

Double replacement of cbh1 and cbh2 with caplacizumab for high productivity and purity. Using T. reesei E1AB1 and E1AB1-XA3 as parental strains, a single replacement of cbh1 and double replacement of cbh1 and cbh2 with a caplacizumab expression cassette that applied the CBD-carrier polypeptide and KEX2 linker (SCK) pattern was performed. The E1AB1 strain and transformants were cultivated in shake flasks on an induction medium containing 3% cellulose, and T. reesei E1AB1-XA3 strain-based transformants were cultivated in shake flasks on a non-induction medium containing 3% glucose. a Total secreted proteins after 2, 3, and 4 days of cultivation. b SDS-PAGE analysis of the secreted proteins, the supernatant after 2 days of cultivation were diluted 4-fold and loaded with 5 µL. Open triangles indicate deleted proteins corresponding to CBH1 and CBH2. The closed triangles indicate caplacizumab. c Concentration of nanobodies secreted into the culture supernatant on days 2 and 4. The concentration of the nanobodies was determined by western blotting using purified caplacizumab as a calibration curve. Error bars indicate standard deviation. Statistical significance was determined using two-tailed unpaired Student’s t-test. *p < 0.05. **p < 0.01