Extracellular production of human hepatitis B virus preS2 antigen as hybrid proteins with Bacillus subtilis ?-amylases in high-salt-concentration media

To produce PreS2 antigen of human hepatitis B virus extra-cellularly in Bacillus subtilis, it was fused with the COOH-termini of B. subtilis alpha-amylases of 522 (Amy+), 467 (Amy+) and 443 (Amy-) amino acids. Among them, alpha-amylase-A467, which has 467 amino acids, was a relatively stable carrier when the cells were cultured in Luria-Bertani (LB) medium. The alpha-amylase-A443-PreS2 hybrid protein (Amy-) was quickly degraded. The alpha-amylase-A522-PreS2 hybrid was most efficiently produced when a B. subtilis transformant of a protease-super-deficient mutant was cultured in the presence of 0.5 M sodium sulphate. The production of A522-PreS2 hybrid protein under such conditions reached 5-10 mg/l and was eight, and 200-500 times higher than those obtained by the transformants of an alkaline/neutral protease-deficient mutant of B. subtilis, and a wild-type strain, in LB medium, respectively.