Extraction and purification of hepatitis B virus-like M particles from a recombinant Saccharomyces cerevisiae strain using alumina powder

• Published in: Journal of virological methods 2013-01, Vol.187 (1), p.132-137
• Main Authors: Hadiji-Abbes, Nadia, Martin, Marta, Benzina, Wafa, Karray-Hakim, Hella, Gergely, Csilla, Gargouri, Ali, Mokdad-Gargouri, Raja
• Format: Article
• Language: English
• Subjects: Aluminum; Aluminum Oxide; Antibodies; Atomic force microscopy; Biological Physics; chronic hepatitis B; double prime M protein; Enzyme-linked immunosorbent assay; Filtration; gel chromatography; glass; Glass beads; Hepatitis B - immunology; Hepatitis B - prevention & control; Hepatitis B Antibodies - blood; Hepatitis B Antibodies - immunology; hepatitis B antigens; Hepatitis B surface antigen; Hepatitis B Surface Antigens - biosynthesis; Hepatitis B Surface Antigens - genetics; Hepatitis B Surface Antigens - immunology; Hepatitis B Vaccines - immunology; Hepatitis B virus; Hepatitis B virus - immunology; Hepatitis B virus - isolation & purification; High-performance liquid chromatography; Humans; Immunoreactivity; Microscopy, Atomic Force; patients; Physics; Powder; proteins; Recombinant M protein Saccharomyces cerevisiae; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Saccharomyces cerevisiae; Saccharomyces cerevisiae - virology; Vaccines; Virus-like particle; Virus-like particles; yeasts
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2012.09.023

► Improvement of HBV M protein extraction by alumina powder from S. cerevisiae. ► Analysis by atomic force microscopy of the rVirus like particle. ► Purification of the rVLP by 2 steps of gel filtration: S-200 and SEC-250. ► Test of the immuno-reactivity of the rVLP on sera of individuals with hepatitis B. A recombinant hepatitis B surface antigen (HBsAg) has been produced in the yeast Saccharomyces cerevisiae 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 (290mg/l) and rVLPs (1mg/l) compared to the values for glass beads (171mg/l and 0.5mg/l), as estimated by quantitative 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.