Purification of a recombinant human respiratory syncytial virus chimeric glycoprotein using reversed-phase chromatography and protein refolding in guanidine hydrochloride.
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, Cu(2+)-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.
Wells
P A
PA
Upjohn Company, Kalamazoo, Michigan 49001.
Garlick
R L
RL
Lyle
S B
SB
Tuls
J L
JL
Poorman
R A
RA
Brideau
R J
RJ
Wathen
M W
MW
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D002845:Chromatography; D005822:Genetic Vectors; D019791:Guanidine; D006146:Guanidines; D015707:HN Protein; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D018357:Respiratory Syncytial Virus Infections / Q000276:immunology / Q000517:prevention & control*; D018113:Respiratory Syncytial Virus, Human; D018411:Spodoptera / Q000166:cytology; D014614:Vaccines, Synthetic / Q000235:genetics / Q000302:isolation & purification*; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification*; D014765:Viral Vaccines / Q000235:genetics / Q000302:isolation & purification*
D006146:Guanidines; D015707:HN Protein; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D014765:Viral Vaccines; C116204:attachment protein G; D019791:Guanidine