Human adenovirus serotype 3 fiber protein. Comparison of native and recombinant proteins.
We were able to isolate viral fiber and penton from Ad3-infected KB cells using for their detection antibodies obtained against recombinant Ad3 fiber. The native material was examined by electron microscopy and the characteristic fiber shape of a shaft terminated by a globular head was observed. The native fiber was compared with two recombinant fibers synthesized in Escherichia coli cells. One, the Ad3 fiber protein expressed in E. coli with a 14-amino acid NH2-terminal fusion peptide, under the control of the T7 promoter has been described previously. The second is a recombinant Ad3 fiber without the fusion peptide (recAd3fib), expressed in the same system. As with the fusion protein recAd3fib was found to be insoluble upon expression. It was solubilized in 6 M urea and the gradual removal of urea during the purification cycle led to a soluble preparation. Biochemical and biophysical studies show that, similarly to fusion fiber, recAd3fib self-assembles as trimers in prokaryotic cells. Electron microscopy shows that, whereas the fusion fiber consists of a population of heterogeneous particles, recAd3fib has the characteristic morphology and size of the Ad3 trimeric native fiber. Small angle neutron scattering gives a molecular weight consistent with a trimeric fiber and a radius of gyration consistent with the dimensions derived from electron microscopy. These results suggest that the fusion peptide at the NH2 terminus prevents correct protein folding. They also indicate that after solubilization with urea and subsequent renaturation a correctly folded eukaryotic oligomeric protein can be produced in E. coli.
Albiges-Rizo
C
C
European Molecular Biology Laboratory, Grenoble Outstation, France.
Barge
A
A
Ruigrok
R W
RW
Timmins
P A
PA
Chroboczek
J
J
D000256:Adenoviridae / Q000378:metabolism*; D015153:Blotting, Western; D002213:Capsid / Q000737:chemistry* / Q000648:ultrastructure; D036022:Capsid Proteins; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D010957:Plasmids; D014764:Viral Proteins / Q000737:chemistry*
D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D014764:Viral Proteins; C018315:hexon capsid protein, Adenovirus