Fig. 2.

Potential improvement of the production host by deletion of vib-1 and introduction of exo-1. Deletion of vib-1 results in a significant decrease in protease activity (a), and the exo-1 mutation causes hypersecretion of a set of proteins (b). To determine protease activity in the culture supernatant (a), the wild-type (WT) and the vib-1 deletion strain (∆vib-1) were cultivated in liquid minimal medium with maltose at 30 °C. Samples of the supernatant were harvested at the indicated time points and incubated for 24 h with 1 µg of HT186-D11. The samples were then analyzed by SDS-PAGE. Presence of the intact antibody fragment is indicated by a band at 30 kD. If protease activity is present in the tested supernatant, smaller degradation products appear or the band fully disappears. To test the hypersecretion of exo-1 strains (b), wild-type (WT) and exo-1 were cultivated for 3 days in liquid minimal medium with maltose at 30 °C. Proteins in the supernatant were precipitated, concentrated (factor 30) and separated with SDS-PAGE. I–III indicates three independent replica. The main band present between 60 and 70 kD represents GLA-1