Purification and characterization of wild-type and ts 112 mutant protein IIIa of human adenovirus 2 expressed in Escherichia coli.
The expression of the protein IIIa gene from human adenovirus type 2 (Ad2) in Escherichia coli has been described previously (M. Cuillel, M. Milleville, and J. C. D'Halluin, 1987, Gene 55, 295-301). The same construct has now been used to express a protein IIIa gene from an Ad2 mutant ts 112 whose functional mutation occurs in this gene. The mutant virus is defective at nonpermissive temperatures in the latest stage of virus maturation. Both the wild-type and ts 112 recombinant proteins are produced in E. coli in an insoluble form, but are readily solubilized in urea. They have the same molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), they sediment as a monomeric species in sucrose gradient centrifugation, and proteolytic digestion reveals a similar pattern for both proteins. Hydrodynamic studies and electron microscopy show that both proteins have an elongated shape, which can be approximated to a cylinder of 20 nm in length and 2.8 nm in diameter. The only well-established difference between the mutant and the wild-type recombinant protein is the higher solubility of the mutant.
Cuillel
M
M
EMBL, Grenoble, France.
Cortolezzis
B
B
Chroboczek
J
J
Langowski
J
J
Ruigrok
R W
RW
Jacrot
B
B
D000260:Adenoviruses, Human / Q000235:genetics*; D036022:Capsid Proteins; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D004589:Electrophoresis, Disc; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D007106:Immune Sera; D015151:Immunoblotting; D008027:Light; D008854:Microscopy, Electron; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D012542:Scattering, Radiation; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure
D036022:Capsid Proteins; D007106:Immune Sera; D011994:Recombinant Proteins; D014764:Viral Proteins; C052227:protein IIIa, Human adenovirus type 2