Rapid purification of monomer HIV-2 Tat protein expressed in Escherichia coli

Human immunodeficiency viruses types 1 and 2 encode transactivator proteins, named Tat-1 and Tat-2, that stimulate transcription directed by the viral long terminal repeat sequences. The Tat-1 and Tat-2 proteins are related in protein sequence and mechanism of transactivation. We expressed Tat proteins by in vitro translation and found, as expected, that both Tat-1 and Tat-2 were monomers. Unexpectedly, we found that the Tat-1 and Tat-2 proteins displayed significantly different tertiary structures. As determined by velocity sedimentation and gel filtration, Tat-1 acted as a compact structure and Tat-2 acted as an extended, asymmetric structure. Additionally, we found that the in vitro-expressed Tat-2 protein was significantly less susceptible to aggregation than the Tat-1 protein. This observation led us to overexpress Tat-2 in Escherichia coli and develop a simple and rapid method to purify monomer Tat-2 to approximately 50% purity. These results suggest that large amounts of Tat-2 protein can be purified in suitable form for biochemical and biophysical studies of Tat protein structure.