Cleavage of procaryotically expressed human immunodeficiency virus fusion proteins by factor Xa and application in western blot (immunoblot) assays.
The proteins p15 and p24 of the human immunodeficiency virus (HIV) type 1 gag gene were expressed as fusion proteins in Escherichia coli for detecting antibodies against the acquired immunodeficiency virus by Western blot (immunoblot) analysis. These fusion proteins contain amino acids 1 to 375 of the E. coli beta-galactosidase linked to the viral protein(s) by a recognition sequence for the specific protease factor Xa. They are obtained in large amounts in insoluble inclusion bodies. To avoid ambiguous results caused by cross-reaction of sera with bacterial proteins in Western blots, we purified the recombinant fusion proteins and subsequently removed the bacterial part of the fusions by cleavage with factor Xa. The cleavage mixtures were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The viral proteins obtained by this method did not contain any bacterial proteins or protein fragments. Thus, false-positive results in HIV Western blot analysis with bacterially expressed HIV proteins can be excluded with these purified recombinant viral antigens.
Ellinger
S
S
Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Federal Republic of Germany.
Glockshuber
R
R
Jahn
G
G
Plückthun
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003429:Cross Reactions; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa; D005786:Gene Expression Regulation; D015683:Gene Products, gag; D005822:Genetic Vectors; D015483:HIV Antibodies / Q000032:analysis*; D015488:HIV Antigens / Q000276:immunology*; D016655:HIV Core Protein p24; D006801:Humans; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D012191:Retroviridae Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D054301:gag Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D015483:HIV Antibodies; D015488:HIV Antigens; D016655:HIV Core Protein p24; D019590:Nucleocapsid Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012191:Retroviridae Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C060295:p15 gag protein, Human immunodeficiency virus 1; D001616:beta-Galactosidase; D012697:Serine Endopeptidases; D015951:Factor Xa