title: Rapid, high-level transient expression of papillomavirus-like particles in insect cells.
abstract: Empirical scanning of natural or engineered peptide sequences for functional residues is inherently dependent upon efficient expression of large numbers of individual sequence variants to assay their relative functional potency. The insect baculovirus system has been widely used for expression of viral coat proteins, but it generally requires prior isolation and expansion of a plaque-purified recombinant viral stock to generate useful quantities of self-assembled virus-like particles. In search of a more rapid means of expression of analytical levels of the L1 coat protein of cottontail rabbit and human type 11 papilloma-viruses, we found that even brief transient cotransfection of insect cells with baculovirus plasmid transfer vectors and viral DNA yielded assembled particles that were immunologically indistinguishable from particles obtained with plaque-purified viral stocks. Within six days of plasmid/viral DNA cotransfection of Sf9 cells, at least 1-2 micrograms of assembled L1 particles/100-mm plate could be demonstrated, which proved more than sufficient to assay functionality. Transient cotransfection of insect cells should provide general utility for rapid high-level expression of sets of protein sequence variants, as well as other sequence-scanning applications such as sequence optimization in protein engineering.
authors.affiliation: Merck Research Laboratories, Rahway, NJ, USA.
authors.forename: D
authors.initials: D
authors.lastname: Benincasa
authors.forename: M
authors.initials: M
authors.lastname: Silberklang
authors.forename: G E
authors.initials: GE
authors.lastname: Mark
authors.forename: S W
authors.initials: SW
authors.lastname: Ludmerer
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D016367:Baculoviridae / Q000235:genetics*; D036022:Capsid Proteins; D002460:Cell Line; D012777:Cottontail rabbit papillomavirus / Q000235:genetics / Q000302:isolation & purification*; D004279:DNA, Viral / Q000032:analysis; D000939:Epitopes; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis; D011817:Rabbits; D018411:Spodoptera / Q000821:virology*; D014162:Transfection
chemical_list: D000914:Antibodies, Viral; D036022:Capsid Proteins; D004279:DNA, Viral; D000939:Epitopes; D009856:Oncogene Proteins, Viral; C063317:L1 protein, Human papillomavirus type 16