Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene

Abstract

An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of I8galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when ,1-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or fl bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of ,l-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified recombinants, and synthesis of H and F proteins was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot detection and Coomassie blue staining. Glycosylation of the proteins appeared to be impaired somewhat, and the precursor to the F protein was not completely cleaved by the proteases present in insect host cells. On the other hand, both proteins appeared to be active in hemagglutination, hemolysis, and cell fusion assays. Levels of synthesis were in the order of 50 to 150 mg of protein per 108 cells.

Purification and characterization of recombinant histidine‐tagged human platelet 12‐lipoxygenase expressed in a baculovirus/insect cell system

“…A tag sequence encoding six histidine residues was introduced by PCR methodology adjacent to the initiation codon for human platelet 12LX cDNA. The full-length, tagged 12LX cDNA was incorporated into the baculovirus transfer vector pETL, which is related to the high-expression vector pJV(Nhe1) [19]. The 124ipoxygenase gene is under the control of the polyhedrin promoter while the ETL promoter' drives j?-galactosidase expression ( Fig.…”

Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E.

“…The triple HA tag (HinDIII-XbaI) encoding the influenza hemagglutinin antigen from the plasmid PACTAG-2 (a gift from A.Charest) was ligated blunt into the SmaI site of KS in front of the p220 sequence to generate KSHAp220. The XbaI (cohesive)-EcoRI (blunt) fragment from the latter construct was subsequently subcloned into the NheI (cohesive)-BamHI (blunt) sites of the plO transfer vector (Vialard et al, 1990).…”

Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

“…Overexpression of MuHN and MeH in S. cerevisiae, as well as MeH in P. pastoris, results in the accumulation of unglycosylated protein precursors within the yeast cytoplasm. Unglycosylated MeH molecules are not detected in the preparations of this protein after synthesis in mammalian expression system [16], however the expression of the MeH protein in insect cells by baculovirus system resulted in a large amount (approximately half of the total product corresponding to 65 kDa species) of unglycosylated MeH protein molecules [31]. The recombinant MeH protein, produced in transgenic carrot plants also had lower molecular weight than the viral protein, suggesting a different glycosylation pattern, but this was not further explored [32].…”