Purification by Two-Phase Partitioning of an Hepatitis Core Protein-Pertussis Epitope Fusion,Expressed in Yeast
It is known that a six amino acid epitope of p69, a membrane protein of Bordetella pertussis, is involved in protection against whooping cough. Chimeric particles were obtained when a thirty amino acid peptide containing the epitope was genetically fused to the hepatitis B virus core antigen and expressed in yeast. The particles were purified by extracting the centrifuged yeast homogenate in a two-phase system composed of PEG 1450 and K phosphate. In this first step, a ten times enrichment was obtained with a total recovery of 45% of the particles. Ninety percent of these were found in the PEG phase. PEG was removed by diafiltration through a YM 100 Amicon membrane with simultaneous concentration of the particles. Gel filtration through a Trisacryl GF 2000 column achieved a good purification and an homogenous preparation as shown by electron microscopy. Immunoaffinity as an alternative to two-phase partitioning will be discussed as well as the advantages of the described protocol for large scale purification of particles.
KeywordsSucrose Gradient Core Particle Bordetella Pertussis Whooping Cough Linear Sucrose Gradient
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