Cloning, expression, purification, and characterization of the major core protein (p26) from equine infectious anemia virus.
The gene coding for the major core protein (p26) of the lentivirus equine infectious anemia virus (EIAV) was cloned from EIAV infected serum, expressed in E. coli, and the resultant protein purified to electrophoretic homogeneity. The protein was expressed in a soluble form and was purified by conventional protein separation methods. When analyzed by SDS-PAGE, under both reducing and non-reducing conditions, the purified protein migrated as a 26 kDa monomer. Recombinant p26 (rp26), therefore, does not contain any intermolecular disulfide bond. Gel filtration chromatography also indicated that the protein occurs as a monomer in solution. Labeling of free sulphydryl groups with [1-14C]iodoacetamide suggests that none of the three cysteine residues of rp26 is involved in intramolecular disulfide bonds. The circular dichroism spectrum of rp26 was consistent with the following assignment of secondary structure elements: 51% a-helix, 15% beta-turn, and 34% aperiodic. Fluorescencespectroscopy revealed that the three tryptophan residues in rp26 occupy two different environments. These data support the conclusion that the recombinant protein is folded into an ordered and probably native conformation. Immunoblotting and enzyme immunoassay with EIAV infected sera demonstrated that recombinant p26 protein may be useful for diagnostic purposes.
Birkett
A J
AJ
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298, USA.
Yélamos
B
B
Rodríguez-Crespo
I
I
Gavilanes
F
F
Peterson
D L
DL
D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D000956:Antigens, Viral / Q000276:immunology; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004859:Equine Infectious Anemia / Q000821:virology; D006736:Horses; D007124:Immunoenzyme Techniques; D004860:Infectious Anemia Virus, Equine / Q000737:chemistry* / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
D000914:Antibodies, Viral; D000956:Antigens, Viral; D011994:Recombinant Proteins; D014758:Viral Core Proteins; C117895:equine infectious anemia virus, p26