title: Purification and functional characterization of wild-type and mutant HIV-1 and HIV-2 Tat proteins expressed in Escherichia coli.
abstract: We describe a single method to purify milligram amounts of authentic wild-type and mutant HIV-1 and HIV-2 Tat proteins overexpressed in Escherichia coli. This method takes advantage of the highly basic, positively charged RNA binding domain present in both HIV-1 and HIV-2 Tat, which also facilitated purification of HIV-1 and HIV-2 mutant Tat proteins. In contrast to previously described methods, our method does not require use of denaturing or reducing agents, since Tat is present in the soluble fraction after bacterial lysis. The activity of purified wild-type and mutant HIV-1 and HIV-2 Tat proteins was determined using cell-based uptake, in vitro transcription, and TAR binding assays. As expected, both HIV-1 and HIV-2 Tat efficiently transactivated the HIV-2 LTR, whereas HIV-2 Tat transactivated the HIV-1 LTR less efficiently than HIV-1 Tat. Purified HIV-2 Tat proteins in which the glutamic acid residue at position 77 was changed to either glycine or glutamine transactivated the HIV-1 LTR more efficiently than wild-type Tat-2, providing additional evidence that the net charge of this region may be responsible for nonreciprocal transactivation between Tat-1 and Tat-2. Our results demonstrate that this method can be used to rapidly purify authentic wild-type and mutant Tat proteins which are suitable for cell-based and in vitro functional studies.
authors.affiliation: Department of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania, 19406, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Orsini
authors.forename: L F
authors.initials: LF
authors.lastname: García-Martínez
authors.forename: G
authors.initials: G
authors.lastname: Mavankal
authors.forename: R B
authors.initials: RB
authors.lastname: Gaynor
authors.forename: C M
authors.initials: CM
authors.lastname: Debouck
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D015696:Gene Products, tat / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017930:Genes, Reporter; D015497:HIV-1 / Q000235:genetics*; D015498:HIV-2 / Q000235:genetics*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010957:Plasmids; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099253:Trans-Activation Responsive RNA-Binding Protein
chemical_list: D015500:Chloramphenicol O-Acetyltransferase; D015696:Gene Products, tat; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099253:Trans-Activation Responsive RNA-Binding Protein