Cloning, high-yield expression in Escherichia coli, and purification of biologically active HIV-1 Tat protein.
We have established an expression system for full-length HIV-1 transactivator (Tat) protein in Escherichia coli. By constructing a synthetic gene for high level expression in enteric bacteria, the recombinant protein can be obtained in high yield. Fusion of the Tat sequence to an N-terminal histidine tag allows the rapid purification of the fusion protein through a single chromatographic step. After cleavage of the fusion protein with CNBr, pure Tat can be obtained through the use of a MonoS column. Reduction of the protein with Tris(2-carboxyethyl)phosphine-HCl and subsequent stepwise refolding yields biologically active Tat. Sample purity and the identity of the protein mass with the mass expected from the amino acid sequence was demonstrated by mass spectrometry. Nuclear magnetic resonance spectroscopy showed the identity of bacterially expressed and chemically synthesized Tat protein (P. Bayer et al., 1995, J. Mol. Biol. 247, 529-535). The expression of Tat in E. coli enables isotope labeling as a prerequisite for multidimensional NMR experiments toward the elucidation of the structure of the Tat-trans-activation response element complex.
Kirsch
T
T
Lehrstuhl für Biopolymere, Universität Bayreuth, Bayreuth, D-95440, Germany.
Boehm
M
M
Schuckert
O
O
Metzger
A U
AU
Willbold
D
D
Frank
R W
RW
Rösch
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D015696:Gene Products, tat / Q000302:isolation & purification*; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D017421:Sequence Analysis; D054322:tat Gene Products, Human Immunodeficiency Virus
D004220:Disulfides; D015696:Gene Products, tat; D011994:Recombinant Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D007544:Isopropyl Thiogalactoside; D008156:Luciferases