High yield expression and purification of HIV-1 Tat(1-72) for structural studies.
High yield expression and purification of HIV-1 Tat(1-72) for structural studies.
J Virol Methods. 2009 Nov 23;
Authors: Shojania S, Henry GD, Chen VC, Vo TN, Perreault H, O'Neil JD
The HIV-1 transactivator of transcription (Tat) is a protein essential for virus replication. Tat is an intrinsically disordered RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains 7 Cys residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purificationof 6xHistidine-tagged and isotopically enriched (in (15)N and (13)C) recombinant HIV-1 Tat(1-72) (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the backbone NMR resonances. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced, monomeric, and unfolded in aqueous solution at pH 4.
PMID: 19941902 [PubMed - as supplied by publisher]
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