Separations for Biotechnology 2 pp 227-237 | Cite as
Purification by Two-Phase Partitioning of an Hepatitis Core Protein-Pertussis Epitope Fusion,Expressed in Yeast
Abstract
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.
Keywords
Sucrose Gradient Core Particle Bordetella Pertussis Whooping Cough Linear Sucrose GradientPreview
Unable to display preview. Download preview PDF.
References
- (1).Lerner, R.A., Green, N., Alexander, H., (1981) Proc. Natl. Acad. Sci. USA 78. 3403–3407.Google Scholar
- (2).Emini, E.A., Jameson, B.A., and Wimmer, E., (1983) Nature 304, 699–703.PubMedCrossRefGoogle Scholar
- (3).Zavala, F., Tam, J.P., Hollingdale, M.R., Cochrane, A.H. Quakyi, I., Nussenzweig, R.S., and Nussenzweig, V., (1985) Science 228, 1436–1440.PubMedCrossRefGoogle Scholar
- (4).Clarke, B.E., Newston, S.E., Carroll, A.R., Francis, M.J., Appleyard, G., Syred, A.D., Highfield, P.E., Rowlands, D.J. and Brown, F., (1987) Nature 330, 381–384.PubMedCrossRefGoogle Scholar
- (5).Milich, D.R., and McLachlan, A., (1986) Science 234 1398–1401.PubMedCrossRefGoogle Scholar
- (6) Mortimer, E.A., (1988) Vaccines 74-79 Ed. Plotkin, S.A. and Mortimer, E.A., Pub. by W.B. Saunders & Co.Google Scholar
- (7).Novotny, P., Kobisch. M., Cownley, K. Chubb, A.P., and Montaraz, J.A., (1985) Infect, and Imnun. 50, 190–198.Google Scholar
- (8).Montaraz, J.A., Novotny, P., and Ivanyi, J., (1985) Infect, and Immun. 467, 744–751.Google Scholar
- (9).Charles, I.G., Dougan, G., Pickard, D., Chatfield, S. Smith, M. Novotny, P., Morrisey, P. and Fairweather N.F., (1989) Proc. Natl. Acad. Sci. 86 3554–3558.PubMedCrossRefGoogle Scholar
- (10).Albertsson, P.A., (1971) Partition of Cell Particles and Macromolecules 2nd Ed. Wiley Interscience.Google Scholar
- (11).Laemmli. U.K., (1970) Nature 227, 680–685.PubMedCrossRefGoogle Scholar
- (12).Nilsson, K., Norrlow, O., and Mosbach, K., (1981) Acta. Chem. Scand. B. 35 19–27.CrossRefGoogle Scholar
- (13).Nilsson, K., and Mosbach, K., (1981) Biochem. Biophys. Res. Commun. 102 449–457.PubMedCrossRefGoogle Scholar
- (14).Beesley, J.E., Day, S.E.J., Betts, M.P. and Thorley, C.M. (1984) J. Gen. Microbiol. 130 1481–1487.PubMedGoogle Scholar
- (15).Serwer, P., (1986). Methods in Enzymology 130, 116-132. Ed. C.H.W. Hirs & S.N.T. Timasheff. Acad. Press Inc.Google Scholar
- (16).Pasek, M., Goto, T, Gilbert, W., Zuik, B., Schaller, H., MacKay, P., Leadbetter, G., and Murray, K. (1979) Nature 282, 575–579.PubMedCrossRefGoogle Scholar
- (17).Argos, P., and Fuller, S.D. (1988) Embo J. 7, 819–824.PubMedGoogle Scholar
- (18).Wasserman, G.F., Inacker, R., Silverman, C.C., and Rosenberg, M., (1987) Protein Purification: Micro to Macro p.337-354. Ed.Alan R. Liss, Inc.Google Scholar
- (19).Stahl, S.J. and Murray, K., (1989) Proc. Natl. Acad. Sci. USA 86, 6283–6287.PubMedCrossRefGoogle Scholar
- (20).Kniskern, P.J., Hagopian, A., Montgomery, D.L., Burke, P., Dunn, N.R Hofman, K.J., Miller, W.J., and Ellis, R.W., (1986) Gene 46 135–141.PubMedCrossRefGoogle Scholar
- (21).Miyanohara, A., Imamura, T., Araki, M. Sugawara, K., Ohtomo, N., and Matsubara, K., (1986) J. Virol. 59 176–180.PubMedGoogle Scholar
- (22).Milich, D.R., McLachlan, A., Stahl, S., Wingfield, P., Thornton, G.B Hughes, J.L., and Jones, J.E. (1988). J,. Immunol. 141, 3617–3624.PubMedGoogle Scholar
- (23).Okada, K., Kamiyama, I., Inomata, M., Imai, M., Miyakawa, Y., and Mayumi, M., (1976) N. Engl. J. Med. 294, 746–749.PubMedCrossRefGoogle Scholar