The study describes expression and purification of recombinant
hepatitis B small
surface antigen (rHBsAg hereafter) in methylotrophic yeast Pichia pastoris strain GS115. For expression of the rHBsAg, a single copy of 678 bp
cDNA was inserted at the unique EcoRI site downstream of the
alcohol oxidase (AOX 1) promoter of the 8.2 kb pHIL-D2 vector. The cDNA-pHIL-D2 construct was used to transform the strain GS115, resulting in a Mut(S) (
Methanol Utilizing Slow) phenotype in which the 226
amino acids containing active and full-length rHBsAg
protein could be expressed intra-cellularly during slow growth and induction with
methanol. The
recombinant protein from the Mut(S) expressor was harvested by cell disruption, and purified first by adsorption-desorption on
aerosil followed by two-step chromatographic separation i.e.
anion exchange on
DEAE resin followed by gel permeation on Superdex 75. Reversed passive hem-agglutination assay (RPHA) was used to test the antigenicity while SDS-PAGE was performed to check the purity of the 27 kDa rHBsAg and its aggregates. The results showed that disruption at 12 Kpsi (three cycles), or 30 Kpsi (1 cycle), desorption with 10mM
carbonate buffer (pH 9-10), and storage at 4 degrees C without
detergent did not adversely affect the antigenicity of the rHbsAg. However, the presence of
detergents such as TritonX100 and
deoxycholate in the disruption and desorption
buffers, respectively resulted in reduced antigenicity during storage both at 4 and -20 degrees C in spite of higher initial yields.