These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Cloning, high-yield expression in Escherichia coli, and purification of biologically active HIV-1 Tat protein.
    Author: Kirsch T, Boehm M, Schuckert O, Metzger AU, Willbold D, Frank RW, Rösch P.
    Journal: Protein Expr Purif; 1996 Aug; 8(1):75-84. PubMed ID: 8812837.
    Abstract:
    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.
    [Abstract] [Full Text] [Related] [New Search]