Fig. 3

Inducer-free nanobodies production by single replacement of cbh1 with three expression patterns of nanobody genes. a Configuration of the nanobody expression cassettes. Each nanobody was combined with the secretory signal peptide of CBH2 (S), the secretory signal peptide and the cellulose-binding domain (CBD) (SC), and the cleavage site of the KEX2 protease between the CBD and the nanobody (SCK). A 6× His-tag sequence was added to the C-terminus of each nanobody. Homologous recombination of the cbh1 locus was performed in the E1AB1-XA3 strain. T. reesei E1AB1-XA3 strain and transformants were cultivated in shake flasks on non-inducing medium containing 3% glucose. b SDS-PAGE analysis of the secreted proteins, the supernatant after 2 days of cultivation were diluted 3-fold and loaded with 5 µL. Open triangles indicate deleted proteins corresponding to CBH1. The closed triangles indicate the target nanobody, as estimated from the molecular weight. c Western blot analysis using an anti-His tag antibody (Fig. 3b). The closed triangles indicate the target nanobody, as estimated from the molecular weight. Dashed triangles indicate cleaved nanobodies. d Concentration of nanobodies secreted into the culture supernatant on day 4. The concentration of the nanobodies was determined by western blotting using the purified nanobody 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