Extraction and purification of hepatitis B virus-like M particles from a recombinant Saccharomyces cerevisiae strain using alumina powder.
Abstract
A recombinant hepatitis B surface antigen (HBsAg) has been produced in the yeast Saccharomyces cere- visiae and used as a vaccine against hepatitis B virus (HBV) infection. The present study aimed to optimize the extraction of recombinant virus-like particles (rVLPs) to develop a simple purification procedure based on gel filtration and high performance size-exclusion chromatography. The findings showed that disruption of yeast cells with alumina powder increased the yield of the total proteins (290 mg/l) and rVLPs (1 mg/l) compared to the values for glass beads (171 mg/l and 0.5 mg/l), as estimated by quantita- tive ELISA. The purification of rVLPs was performed by two consecutive gel filtration chromatographic steps, namely Sephacryl S-200 followed by SEC-250 HPLC. The purified M protein was analyzed by atomic force microscopy and shown to assemble in particles that were able to recognize HBV antibodies in the sera of patients with chronic hepatitis B, providing evidence for their immunoreactivity.