| 초록 |
Here, we describe the purification of recombinant HBsAg from P. pastoris GS115 (Muts). The 678 bp fragment encoding surface antigen S protein was inserted at the EcoR I site downstream of the AOX1 promoter of the pHIL-D2 vector. The 226 amino acid containing HBsAg expressed intra-cellularly during induction with methanol was purified by adsorption-desorption on aerosil followed by chromatographic separation on DEAE resin and gel permeation using Superdex 75. Reverse passive hemagglutination was used for the functional analysis. While the purity of 27 kDa HBsAg was tested with SDS-PAGE and Western blot. Additionally, effect of detergent on extraction, disruption pressure, storage temperature, adsorption time on aerosil, and composition of desorption buffer were studied. In general, with detergents, the total protein yield was higher, but greater loss of HBsAg activity subsequently during storage at 4°C was also recorded. Nevertheless, disruption at 12 Kpsi (3 cycles), or 30 Kpsi (1cycle), desorption with 10 mM carbonate buffer (pH 9-10), and storage at 4°C (without detergent) were beneficial. |
