Secretion and purification of HCV E1 protein forms as glutathione-S-transferase fusion in the baculovirus insect cell system.
We have expressed the E1 protein of Hepatitis C Virus (HCV) in a new recombinant form by using a baculovirus transfer vector directing the expression of proteins fused to the carboxy-terminus of glutathione-S-transferase (GST). The E1 domain was expressed varying at its carboxy terminus in order to retain (GST-E1) or delete (GST-E1b) the C-terminal hydrophobic region that may be involved in membrane association. Following infection with the recombinant virus, GST-E1b was efficiently secreted into the culture media and could be purified in a single step with the minimum of denaturation by glutathione affinity chromatography. The purified product was specifically immunoprecipitated by HCV positive human sera suggesting the maintenance of an immuno-relevant tertiary structure despite removal of the hydrophobic anchor. By contrast, cells infected with a recombinant baculovirus expressing GST-E1 gave a fusion protein with an appropriate molecular weight but also a series of polypeptides of lower molecular weight consistent with cleavage at the C-terminus of E1. GST-E1 was not secreted into the medium and was associated predominantly with the membrane fraction following cell disruption; the lower molecular weight forms were soluble and secreted.
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Laboratory of Virology, Istituto Superiore di Sanità, Rome, Italy. equestre@virus1.net.iss.it
Marcantonio
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Equestre
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Jones
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Rapicetta
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D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D003001:Cloning, Molecular; D003470:Culture Media; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D018411:Spodoptera; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D003470:Culture Media; C087035:E1 protein, Hepatitis C virus; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D005982:Glutathione Transferase; D006596:Hexosaminidases