EXPRESSION OF CHICK AND YEAST BETA-TUBULIN-ENCODING GENES IN INSECT CELLS

A chick cDNA encoding the beta2 isotype of tubulin (beta2Tub) was cloned into a baculovirus expression vector designed to produce unfused proteins, and several recombinant viruses (re-viruses) were isolated. Immunoblotting studies of homogenates of insect cells infected with re-virus showed a 50-kDa protein that reacted with antibodies specific for betaTub. Cells infected with the re-virus appeared to contain much higher levels of betaTub than uninfected control cells, perhaps as much as five- to tenfold higher. Isotype-specific antibody for beta2Tub showed little reaction in uninfected cells or cells infected with wild-type virus; strong reaction was found with cells infected with re-virus. Analysis by gel filtration of extracts of cells infected with re-virus showed that almost all betaTub eluted in the column void volume, suggesting that it was aggregated or associated with other cell proteins. Recombinant baculoviruses producing Saccharomyces cerevisiae betaTub were also isolated. Immunoblotting studies using antibodies specific for yeast betaTub showed a 50-kDa protein which was absent in uninfected cells or cells infected with wt virus. Immunofluorescence studies suggest that yeast betaTub is incorporated poorly, if at all, into the insect cell cytoskeleton.