Expression of bovine leukemia virus ENV glycoprotein in insect cells by recombinant baculovirus

• Published in: FEBS letters Vol. 436; no. 1; pp. 11 - 16
• Main Authors: Russo, Silvia, Montermini, Laura, Berkovitz-Siman-Tov, Revital, Ponti, Wilma, Poli, Giorgio
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 25.09.1998
• Subjects: AcMNPV, Autographa californica multiple nuclear polyhedrosis virus; Animals; Baculoviridae - genetics; Baculovirus; BCA, bichinchoninic acid protein quantitative assay; BEVS, baculovirus expression vector system; Biotechnology; Blotting, Western; BLV, bovine leukemia virus; Bovine leukemia virus; bp, base pair; BSA, bovine serum albumin; Carbohydrate Sequence; DEPC, diethyl-pyrocarbonate; FCS, fetal calf serum; FLK, fetal lamb kidney cell line; FLK-gp51, gp51 purified from persistently infected FLK cell; Glycosylation; gp51-p30, glycoproteins constituting BLV envelope; Inclusion Bodies - chemistry; Insecta - virology; kbp, 103 base pairs; kD, 103 daltons; Kinetics; Lectins - metabolism; MAb, monoclonal antibody; MOI, multiplicity of infection; Molecular Sequence Data; occ −, AcMNPV plaques showing polh− phenotype; p.i., post infection; PBS, phosphate buffered saline; PCR, polymerase chain reaction; polh, polyhedrin coding gene of AcMNPV; PRINS, primed in situ labelling of nucleic acids; Protein Engineering - methods; Recombinant glycoprotein; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Retroviridae Proteins, Oncogenic - genetics; Retroviridae Proteins, Oncogenic - isolation & purification; Retroviridae Proteins, Oncogenic - metabolism; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; Solubility; Substrate Specificity; Viral Envelope Proteins - genetics; Viral Envelope Proteins - isolation & purification; Viral Envelope Proteins - metabolism; Biotechnology; occ −, AcMNPV plaques showing polh − phenotype; Baculovirus; BLV, bovine leukemia virus; polh, polyhedrin coding gene of AcMNPV; bp, base pair; FLK, fetal lamb kidney cell line; BEVS, baculovirus expression vector system; gp51-p30, glycoproteins constituting BLV envelope; PRINS, primed in situ labelling of nucleic acids; kbp, 10 3 base pairs; BSA, bovine serum albumin; Recombinant glycoprotein; p.i., post infection; PBS, phosphate buffered saline; Glycosylation; Bovine leukemia virus; DEPC, diethyl-pyrocarbonate; AcMNPV, Autographa californica multiple nuclear polyhedrosis virus; MAb, monoclonal antibody; MOI, multiplicity of infection; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; BCA, bichinchoninic acid protein quantitative assay; PCR, polymerase chain reaction; kD, 10 3 daltons; FCS, fetal calf serum; FLK-gp51, gp51 purified from persistently infected FLK cell
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(98)00951-X

The gp51-p30 glycoprotein constituting BLV envelope was expressed in Sf-21 insect cells by means of recombinant baculoviruses. Post-infection cell lysates were analyzed, in order to define the immunologic reactivity of recombinant products. Oligosaccharide chains, containing N-acetylglucosamine, mannose, galactose and sialic acid were found on recombinant gp51-p30. In order to investigate the timing of transcription and translation of the glycoprotein, kinetic assays were carried out on cell lysates and directly in situ on Sf-21 cells during the course of baculovirus infection. The use of different solubilizing reagents was also evaluated in order to rescue recombinant glycoprotein from its subcellular location.