We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
Translational incorporation ofL-3,4-dihydroxyphenylalanine into proteins.
- Authors
Ozawa, Kiyoshi; Headlam, Madeleine J.; Mouradov, Dmitri; Watt, Stephen J.; Beck, Jennifer L.; Rodgers, Kenneth J.; Dean, Roger T.; Huber, Thomas; Otting, Gottfried; Dixon, Nicholas E.
- Abstract
An Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation ofl-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean15N-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell-free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and/or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome-mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High-yield cell-free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.
- Subjects
DOPA; CATECHOLAMINES; PROTEINS; ESCHERICHIA coli; TYROSINE; PROTEIN folding
- Publication
FEBS Journal, 2005, Vol 272, Issue 12, p3162
- ISSN
1742-464X
- Publication type
Article
- DOI
10.1111/j.1742-4658.2005.04735.x