Structural proteins of adenovirus. Expression in Escherichia coli.
The fiber proteins of adenovirus serotype 2 Ad2 and serotype 3 Ad3 and structural protein IIIa of wild type Ad2 and Ad2 ts 112 mutant were cloned and expressed in E. coli. For the expression of both fiber proteins a gene expression system based on bacteriophage T7 RNA polymerase was used. The expressed proteins constituted 1-3% of total host cell protein. Both proteins were insoluble and inclusion bodies were observed. The proteins could be purified from cellular debris by extraction with 6 M urea followed by chromatography in the presence of diminishing concentration of urea. The folding of recombinant fiber proteins was assessed by sensitivity to proteases and gel filtration. Both proteins were synthetized as trimers. Ad2 recombinant fiber has a much less compact structure than native Ad2 fiber, since on gel filtration it is excluded before the native fiber. It is also much more sensitive to chymotrypsin digestion than the native protein. Contrary to that, Ad3 recombinant fiber is much less sensitive to proteolytic cleavage and on gel filtration has the same exclusion volume as the trimeric native fiber of Ad3.
Chroboczek
J
J
European Molecular Biology Laboratory, Grenoble Outstation, France.
Chatellard
C
C
Rizo
C
C
Cuillel
M
M
Jacrot
B
B
D000260:Adenoviruses, Human / Q000235:genetics*; D015153:Blotting, Western; D036022:Capsid Proteins; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005814:Genes, Viral; D006801:Humans; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification; D013604:T-Phages / Q000201:enzymology; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification; D015678:Viral Structural Proteins / Q000235:genetics*
D036022:Capsid Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C052227:protein IIIa, Human adenovirus type 2; D012321:DNA-Directed RNA Polymerases