title: Rapid purification of monomer HIV-2 Tat protein expressed in Escherichia coli.
abstract: 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.
authors.affiliation: Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: H
authors.initials: H
authors.lastname: Rhim
authors.forename: F
authors.initials: F
authors.lastname: Chan
authors.forename: C O
authors.initials: CO
authors.lastname: Echetebu
authors.forename: A P
authors.initials: AP
authors.lastname: Rice
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015696:Gene Products, tat / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015498:HIV-2 / Q000235:genetics*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus