Secretion and purification of HCV E1 protein forms as glutathione- S-transferase fusion in the baculovirus insect cell system

• Published in: Virus research Vol. 55; no. 2; pp. 157 - 165
• Main Authors: Ciccaglione, Anna R, Marcantonio, Cinzia, Equestre, Michele, Jones, Ian M, Rapicetta, Maria
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.1998
• Subjects: Animals; Baculoviridae; Baculovirus; Cell Line; Cloning, Molecular; Culture Media; Envelope protein; ER retention signal; Gene Expression; Genetic Vectors; Glutathione Transferase - genetics; HCV; Hepacivirus - genetics; Hepacivirus - metabolism; Hexosaminidases - metabolism; Humans; Precipitin Tests; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Secretion; Solubility; Spodoptera; Viral Envelope Proteins - biosynthesis; Viral Envelope Proteins - genetics; Viral Envelope Proteins - isolation & purification; Envelope protein; HCV; Baculovirus; ER retention signal; Secretion
• ISSN: 0168-1702; 1872-7492
• DOI: 10.1016/S0168-1702(98)00041-0

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.