Recovery and characterization of a rotavirus outer capsid protein expressed in a recombinant insect cell system.
Recombinant OSU VP4 protein, and outer capsid antigen of porcine rotavirus, was purified to a high level from the spent broth of baculovirus-infected Spodoptera frugiperda insect cells. Initial clarification of the broth with a 0-60% ammonium sulfate cut retained 93% of the total VP4. Q-sepharose ion exchange chromatography performed at pH 6.5 yielded 67% of the initial amount of VP4 in the pooled fractions, with more than four times the purity of the original sample. Gel filtration chromatography followed ion exchange. VP4 eluted from this column at a volume corresponding to a protein of a molecular weight of approximately 85 kDa, the single chain molecular weight of VP4. This step retained 34% of the initial VP4, with a 28-fold purification. Affinity chromatography, using heparin and glycophorin as ligands, was chosen as a final polishing step. The selective binding of VP4 to the two ligands suggested that VP4 may play a role during in vivo rotaviral infection. These specific interactions based on the biological properties of rotaviruses achieved a VP4 purity level of 85-95%. The overall purification scheme recovered about 1.5 mg per liter of VP4 spent broth from about 50 mg/liter (5% of total protein) present initially in the broth.
Department of Chemical Engineering, Johns Hopkins University, Baltimore, Maryland 21218.
L G
LG
Juarbe-Osorio
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Gorziglia
M J
MJ
Betenbaugh
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002213:Capsid / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D036022:Capsid Proteins; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D009036:Moths / Q000166:cytology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012401:Rotavirus / Q000235:genetics*; D013552:Swine / Q000382:microbiology
D036022:Capsid Proteins; D011994:Recombinant Proteins; C060339:VP4 protein, Rotavirus