Expression and purification of recombinant HIV-1 tat protein using HIV-1-tat specific monoclonal antibodies.
We expressed tat protein from HIV-1 coding AA 1-67 (strain HIV-Bru) in the inducible vector pTrc 99A in E. coli. The tat coding region was cloned in the ATG site of the expression vector. A sequence coding for 15 AA was added at the 3'region as a molecular marker. After sonification, the tat protein was routinely detected in Western blots using the Mab developed by us. Following precipitation and centrifugation the resulting pellets were dissolved and purified by three different methods: 1. immunoaffinity-chromatography using Affi-gel HZ coupled with a Mab recognizing the N-terminal sequence of HIV-1-tat; 2. ion exchange chromatography using DEAE-52 cellulose, and 3. isoelectric focusing in free solution. The resulting protein extracts obtained from the three purification protocols were checked in ELISA with the antibody. The peak fraction from all the procedures showed tat activity. No cross reaction in the presence of sera from uninfected persons was observed. The results showed that the purification of tat protein using monoclonal antibodies leads to highly purified preparations.
Demirhan
I
I
Department of Molecular Biology (ZBC), Frankfurt University Medical School, Germany.
Hofmann
D
D
Chandra
A
A
Hasselmayer
O
O
Chandra
P
P
D000911:Antibodies, Monoclonal / Q000276:immunology*; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D015696:Gene Products, tat / Q000096:biosynthesis* / Q000302:isolation & purification; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D054322:tat Gene Products, Human Immunodeficiency Virus
D000911:Antibodies, Monoclonal; D015696:Gene Products, tat; D011994:Recombinant Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus