Expression and purification of recombinant HIV-1 tat protein using HTV-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 t he 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. Fol lowing precipitation and centrifugation the resulting pellets were dissolve d and purified by three different methods: 1. immunoaffinity-chromatography using Affi-gel HZ coupled with a Mab recognizing the N-terminal sequence o f HIV-1-tat; 2. ion exchange chromatography using DEAE-52 cellulose, and 3. isoelectric focusing in free solution. The resulting protein extracts obta ined from the three purification protocols were checked in ELISA with the a ntibody. 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 rat protein using monoclonal antibodies leads to highly purified preparations.