Purification of a Recombinant Human Respiratory Syncytial Virus Chimeric Glycoprotein Using Reversed-Phase Chromatography and Protein Refolding in Guanidine Hydrochloride
Identifieur interne : 004404 ( Main/Exploration ); précédent : 004403; suivant : 004405Purification of a Recombinant Human Respiratory Syncytial Virus Chimeric Glycoprotein Using Reversed-Phase Chromatography and Protein Refolding in Guanidine Hydrochloride
Auteurs : P. A. Wells [États-Unis] ; R. L. Garlick [États-Unis] ; S. B. Lyle [États-Unis] ; J. L. Tuls [États-Unis] ; R. A. Poorman [États-Unis] ; R. J. Brideau [États-Unis] ; M. W. Wathen [États-Unis]Source :
- Protein Expression and Purification [ 1046-5928 ] ; 1994.
Abstract
Abstract: FG glycoprotein is a recombinant chimeric protein consisting of the extracellular portions of human respiratory syncytial virus (RSV) F and G glycoproteins. In theory, highly purified FG glycoprotein may be effective as a RSV vaccine. Recombinant FG glycoprotein was expressed using the baculovirus/insect cell system. FG glycoprotein was isolated from cell culture supernatants using S Sepharose ion-exchange chromatography, Cu2+-immobilized metal affinity chromatography, preparative reversed-phase high-performance liquid chromatography, denaturation with 6 M guanidine hydrochloride, and protein refolding in Tween 80 detergent. The purified FG glycoprotein was concentrated on a S Sepharose column and exchanged into an appropriate buffer for vaccine formulation. Five batches of FG glycoprotein with protein purity of 92-99% were produced using this purification process. FG glycoprotein produced using reversed-phase chromatography and protein refolding was compared with nondenatured FG glycoprotein using a panel of 14 monoclonal antibodies directed against conformational and linear epitopes on RSV F and G glycoproteins. The results of these studies indicated that refolded FG glycoprotein had the same three-dimensional structure as nondenatured FG glycoprotein.
Url:
DOI: 10.1006/prep.1994.1057
Affiliations:
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In theory, highly purified FG glycoprotein may be effective as a RSV vaccine. Recombinant FG glycoprotein was expressed using the baculovirus/insect cell system. FG glycoprotein was isolated from cell culture supernatants using S Sepharose ion-exchange chromatography, Cu2+-immobilized metal affinity chromatography, preparative reversed-phase high-performance liquid chromatography, denaturation with 6 M guanidine hydrochloride, and protein refolding in Tween 80 detergent. The purified FG glycoprotein was concentrated on a S Sepharose column and exchanged into an appropriate buffer for vaccine formulation. Five batches of FG glycoprotein with protein purity of 92-99% were produced using this purification process. FG glycoprotein produced using reversed-phase chromatography and protein refolding was compared with nondenatured FG glycoprotein using a panel of 14 monoclonal antibodies directed against conformational and linear epitopes on RSV F and G glycoproteins. The results of these studies indicated that refolded FG glycoprotein had the same three-dimensional structure as nondenatured FG glycoprotein.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country></list><tree><country name="États-Unis"><noRegion><name sortKey="Wells, P A" sort="Wells, P A" uniqKey="Wells P" first="P. A." last="Wells">P. A. Wells</name></noRegion><name sortKey="Brideau, R J" sort="Brideau, R J" uniqKey="Brideau R" first="R. J." last="Brideau">R. J. Brideau</name><name sortKey="Garlick, R L" sort="Garlick, R L" uniqKey="Garlick R" first="R. L." last="Garlick">R. L. Garlick</name><name sortKey="Lyle, S B" sort="Lyle, S B" uniqKey="Lyle S" first="S. B." last="Lyle">S. B. Lyle</name><name sortKey="Poorman, R A" sort="Poorman, R A" uniqKey="Poorman R" first="R. A." last="Poorman">R. A. Poorman</name><name sortKey="Tuls, J L" sort="Tuls, J L" uniqKey="Tuls J" first="J. L." last="Tuls">J. L. Tuls</name><name sortKey="Wathen, M W" sort="Wathen, M W" uniqKey="Wathen M" first="M. W." last="Wathen">M. W. Wathen</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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