Overexpression and purification of an immunologically reactive His-BIV capsid fusion protein.
The gene of the capsid protein of bovine immunodeficiency virus (BIV) was linked to a sequence encoding for six histidines and expressed as the (His)6 p26 capsid fusion protein. The fusion protein was strongly expressed as both soluble and insoluble forms after induction by isopropylthio-beta-d-galactoside. Purification was based on interaction of the hexa-histidine polypeptide with metal ions. Expression could represent 11% of the total protein in Escherichia coli, allowing more than 20 mg of highly purified protein to be obtained per liter of bacterial culture. The (His)6 p26 capsid fusion protein purified by immobilized metal affinity chromatography reacted specifically in Western blot with sera from cattle experimentally infected by BIV, as well as with two monoclonal antibodies directed against different epitopes of the Gag protein. The ease of expression, purification, and specificity of this fusion protein should permit a thorough study of prevalence of BIV infection in large-scale serological studies of field samples.
Betemps
D
D
Centre National d'Etudes Vétérinaires et Alimentaires, Lyon, France.
Mallet
F
F
Cheynet
V
V
Baron
T
T
D000818:Animals; D000911:Antibodies, Monoclonal; D002213:Capsid / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015683:Gene Products, gag / Q000276:immunology; D006639:Histidine; D016087:Immunodeficiency Virus, Bovine / Q000235:genetics* / Q000378:metabolism; D007544:Isopropyl Thiogalactoside; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D012685:Sepharose
D000911:Antibodies, Monoclonal; D015683:Gene Products, gag; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D006639:Histidine; D012685:Sepharose