Purification and in vitro-phospholabeling of secretory envelope proteins E1 and E2 of hepatitis C virus expressed in insect cells.
The putative envelope glycoproteins of hepatitis C virus (HCV), E1 and E2, were expressed as recombinant, secretory proteins in Sf9 insect cells through infection with recombinant baculoviruses. The influenza virus hemagglutinin signal sequence (HASS) was inserted upstream of the HCV-cDNAs in order to effect secretion. Furthermore, a hexa-histidine tag for purification on a Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) column and a protein kinase A (PKA) recognition sequence for in vitro-phospholabeling were fused upstream of the HCV-cDNA. E1- and E2 proteins lacking their carboxy-terminal, hydrophobic sequence were produced by baculovirus-infected insect cells in bioreactors of 23 1. The medium was concentrated and proteins were purified under native conditions on Ni(2+)-NTA columns. Purified proteins could be phospholabeled in vitro using the catalytic subunit of protein kinase. A isolated from bovine heart and gamma-[32P]ATP. Labeled E1 and E2 proteins expressed in insect cells could be immunoprecipitated with sera from HCV-infected patients. Co-expression of these E1 and E2 proteins led to the formation of E1-E2 complexes within the insect cell and to secretion of these complexes into the medium.
Department of a Pharmaceutical Research-Gene Technology, Basel, Switzerland. Peter.Huessy@Roche.com
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Hüssy
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Schmid
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Mous
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Jacobsen
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D016367:Baculoviridae; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D046508:Culture Techniques / Q000379:methods; D017868:Cyclic AMP-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006526:Hepatitis C / Q000097:blood / Q000276:immunology; D018937:Hepatitis C Antibodies / Q000097:blood; D006639:Histidine; D006801:Humans; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011865:Radioisotope Dilution Technique; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D018411:Spodoptera; D014162:Transfection; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*
D017931:DNA Primers; C087035:E1 protein, Hepatitis C virus; D018937:Hepatitis C Antibodies; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus; D006639:Histidine; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases