Exploiting the intracellular compartmentalization characteristics of the S. cerevisiae host cell for enhancing primary purification of lipid-envelope virus-like particles.
This article demonstrates how the intracellular compartmentalization of the S. cerevisiae host cell can be exploited to impart selectivity during the primary purification of lipid-envelope virus-like particles (VLPs). The hepatitis B surface antigen (HBsAg) was used as the VLP model in this study. Expressed HBsAg remain localized on the endoplasmic reticulum and the recovery process involves treating cell homogenate with a detergent for HBsAg liberation. In our proposed strategy, a centrifugation step is introduced immediately following cell disruption but prior to the addition of detergent to allow the elimination of bulk cytosolic contaminants in the supernatant, achieving approximately 70% reduction of contaminating yeast proteins, lipids, and nucleic acids. Recovery and subsequent treatment of the solids fraction with detergent then releases the HBsAg into a significantly enriched product stream with a yield of approximately 80%. The selectivity of this approach is further enhanced by operating under moderate homogenization pressure conditions ( approximately 400 bar). Observed improvements in the recovery of active HBsAg and reduction of contaminating host lipids were attributed to the low-shear conditions experienced by the HBsAg product and reduced cell fragmentation, which led to lower coextraction of lipids during the detergent step. As a result of the cleaner process stream, the level of product capture during the loading stage of a downstream hydrophobic interaction chromatography stage increased by two-fold leading to a concomitant increase in the chromatography step yield. The lower level of exposure to contaminants is also expected to improve column integrity and lifespan.
Kee
Gaik Sui
GS
Dept. of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K.
Jin
Jing
J
Balasundaram
Bangaru
B
Bracewell
Daniel G
DG
Pujar
Narahari S
NS
Titchener-Hooker
Nigel J
NJ
D002451:Cell Compartmentation; D002478:Cells, Cultured; D005591:Chemical Fractionation; D003902:Detergents / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006514:Hepatitis B Surface Antigens / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism / Q000821:virology*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D003902:Detergents; D006514:Hepatitis B Surface Antigens; C118669:S envelope protein, hepatitis B virus; D014759:Viral Envelope Proteins