Expression of chick and yeast beta-tubulin-encoding genes in insect cells.
A chick cDNA encoding the beta 2 isotype of tubulin (beta 2Tub) 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 beta Tub. Cells infected with the re-virus appeared to contain much higher levels of beta Tub than uninfected control cells, perhaps as much as five- to tenfold higher. Isotype-specific antibody for beta 2Tub 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 beta Tub eluted in the column void volume, suggesting that it was aggregated or associated with other cell proteins. Recombinant baculoviruses producing Saccharomyces cerevisiae beta Tub were also isolated. Immunoblotting studies using antibodies specific for yeast beta Tub showed a 50-kDa protein which was absent in uninfected cells or cells infected with wt virus. Immunofluorescence studies suggest that yeast beta Tub is incorporated poorly, if at all, into the insect cell cytoskeleton.
Vats-Mehta
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S
Department of Biochemistry, University of Kansas Medical Center, Kansas City 66160-7421.
Yarbrough
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LR
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002645:Chickens / Q000235:genetics; D002850:Chromatography, Gel; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000648:ultrastructure; D009036:Moths / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013045:Species Specificity; D014162:Transfection; D014404:Tubulin / Q000235:genetics*
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D014404:Tubulin