Purification of an active monomeric recombinant HIV-1 trans-activator.
A method for the purification of a truncated, biologically active human immunodeficiency virus type 1 (HIV-1) trans-activator (rTAT) from recombinant Escherichia coli is reported here. The purification steps utilized include mild extraction (French press), concentration by ammonium sulfate precipitation, chromatography in 8 M urea on an S-Sepharose fast-protein liquid chromatography column, and finally, resolution by C-4 reverse-phase high-performance liquid chromatography. After the final step, the rTAT is dried and stored under salt-free conditions. Amino acid compositional analysis and N-terminal sequence analysis confirm that the purified protein is rTAT. Unlike other methods reported for purification of recombinant HIV-1 trans-activator, our protocol uses urea instead of guanidine HCl. The rTAT is fully soluble in buffered solutions at concentrations exceeding 10 mg/ml, migrates as a single 14 kDa species on both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and two-dimensional PAGE gels with a pI of 9.3 +/- 0.3. Additionally, the rTAT migrates as a monomer on size-exclusion chromatography columns under native conditions. Finally, purified rTAT exhibits trans-activator activity when introduced into appropriate reporter cells. Since rTAT is monomeric when tested by gel filtration, and yet exhibits biological activity, we conclude that the method of purification we have utilized is distinct from all other methods reported to date.
Waszak
G A
GA
Upjohn Laboratories, Upjohn Company, Kalamazoo, Michigan 49001.
Hasler
J M
JM
McQuade
T J
TJ
Tarpley
W G
WG
Deibel
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002847:Chromatography, Agarose; D004285:Dogs; D004926:Escherichia coli; D015696:Gene Products, tat / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D015497:HIV-1 / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D015533:Transcriptional Activation; D014508:Urea; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D014508:Urea