Purification and characterization of pre-S-containing hepatitis B surface antigens produced in recombinant mammalian cell culture.
Heterogeneous, pre-S-rich HBsAg particles were expressed in recombinant mammalian cell culture and purified to near homogeneity. The purification process comprises: concentration of cell culture medium, protein precipitation by poly(ethylene glycol), gel filtration column chromatography, isopycnic ultracentrifugation by KBr and sucrose density gradient ultracentrifugation. The resulting HBsAg product was greater than 98% pure, and contained much of pre-S1 and pre-S2 components. Scanning densitometry analysis of the silver-stained HBsAg product showed approximately 70-80% S protein, approximately 10-20% pre-S2 protein, and approximately 5-15% pre-S1 protein. It was estimated that the amount of HBV-specific DNA present the final product was less than 7 pg mg-1 HBsAg. Further biochemical analysis has demonstrated that the HBsAg particles are very heterogeneous in charge and density. Charge heterogeneity was quite random among the particles, but density heterogeneity could be related to the different amounts of pre-S2 component in the particles.
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Eugene Tech International, Allendale, NJ 07401.
Samanta
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D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005796:Genes; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011498:Protein Precursors / Q000302:isolation & purification; D011995:Recombination, Genetic; D012967:Sodium Dodecyl Sulfate
D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D012967:Sodium Dodecyl Sulfate