Structure and expression in E. coli of the gene coding for protein p10 of African swine fever virus.
The gene encoding protein p10, a structural protein of African swine fever (ASF) virus, has been mapped, sequenced and expressed in E. coli. Protein p10 was purified from dissociated virus by reverse-phase HPLC, and its NH2-terminal end identified by automated Edman degradation. To map the gene encoding protein p10, a mixture of 20-mer oligonucleotides based upon a part of the amino acid sequence was hybridized to cloned ASF virus restriction fragments. This allowed the localization of the gene in fragment Eco RI K of the ASF virus genome. The nucleotide sequence obtained from this region revealed an open reading frame encoding 78 amino acids, with a high content of Ser and Lys residues. Several of the Ser residues are found in Ser-rich regions, which are also found in some nucleic acid-binding proteins. The gene coding for protein p10 has been inserted in an expression vector which contains the promoter for T7 RNA polymerase. The recombinant plasmid was used to produce the ASF virus protein in E. coli. The bacterially produced p10 protein shows a strong DNA binding activity with similar affinity for both double-stranded and single-stranded DNA.
Departamento de Biología Funcional Facultad de Medicina, Universidad de Oviedo, Spain.
M
M
Muñoz
J M
JM
Freije
M L
ML
Salas
E
E
Viñuela
C
C
López-Otín
D000358:African Swine Fever Virus / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005814:Genes, Viral; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014709:Vero Cells; D015678:Viral Structural Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; C081406:protein p10, African swine fever virus