High yield expression and purification of HIV-1 Tat1-72 for structural studies.
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 seven 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 purification of 6xHistidine-tagged and isotopically enriched (in N15 and C13) 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.
Shojania
Shaheen
S
Department of Chemistry, University of Manitoba, Winnipeg, MB R3T 2N2, Canada. shojania@gmail.com
Henry
Gillian D
GD
Chen
Vincent C
VC
Vo
Thach N
TN
Perreault
Hélène
H
O'Neil
Joe D
JD
D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D013058:Mass Spectrometry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; C470431:tat peptide (1-72), Human immunodeficiency virus 1