at Google ScholarTitle data
Kirsch, Thomas ; Böhm, Markus ; Schuckert, Oliver ; Metzger, Armin U. ; Willbold, Dieter ; Frank, Rainer W. ; Rösch, Paul:
Cloning, High-Yield Expression in Escherichia coli, and Purification of Biologically Active HIV-1 Tat Protein.
In: Protein Expression and Purification.
Vol. 8
(1996)
Issue 1
.
- pp. 75-84.
ISSN 1046-5928
DOI: https://doi.org/10.1006/prep.1996.0076
Abstract in another language
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.
Further data
| Item Type: | Article in a journal |
|---|---|
| Refereed: | Yes |
| Institutions of the University: | Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors > Chair Biopolymers - Univ.-Prof. Dr. Paul Rösch Faculties Faculties > Faculty of Biology, Chemistry and Earth Sciences Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry with an Emphasis on Biophysical Chemistry |
| Result of work at the UBT: | Yes |
| DDC Subjects: | 500 Science > 540 Chemistry 500 Science > 570 Life sciences, biology |
| Date Deposited: | 11 Jan 2019 07:58 |
| Last Modified: | 31 Mar 2022 13:34 |
| URI: | https://eref.uni-bayreuth.de/id/eprint/46881 |