Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins

Ozawa, K; Headlam, MJ; Mouradov, D; Watt, SJ; Beck, JL; Rodgers, KJ; Dean, RT; Huber, T; Otting, G; Dixon, NE

Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins

Ozawa, K Headlam, MJ Mouradov, D Watt, SJ Beck, JL Rodgers, KJ

Dean, RT Huber, T Otting, G Dixon, NE

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Publication Type:: Journal Article
Citation:: FEBS Journal, 2005, 272 (12), pp. 3162 - 3171
Issue Date:: 2005-06-01

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Field	Value	Language
dc.contributor.author	Ozawa, K	en_US
dc.contributor.author	Headlam, MJ	en_US
dc.contributor.author	Mouradov, D	en_US
dc.contributor.author	Watt, SJ	en_US
dc.contributor.author	Beck, JL	en_US
dc.contributor.author	Rodgers, KJ https://orcid.org/0000-0002-3627-9651	en_US
dc.contributor.author	Dean, RT	en_US
dc.contributor.author	Huber, T	en_US
dc.contributor.author	Otting, G	en_US
dc.contributor.author	Dixon, NE	en_US
dc.date.issued	2005-06-01	en_US
dc.identifier.citation	FEBS Journal, 2005, 272 (12), pp. 3162 - 3171	en_US
dc.identifier.issn	1742-464X	en_US
dc.identifier.uri	http://hdl.handle.net/10453/14959
dc.description.abstract	An Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation of L-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 clean 15N-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. © 2005 FEBS.	en_US
dc.relation.ispartof	FEBS Journal	en_US
dc.relation.isbasedon	10.1111/j.1742-4658.2005.04735.x	en_US
dc.subject.classification	Biochemistry & Molecular Biology	en_US
dc.subject.mesh	Cell-Free System	en_US
dc.subject.mesh	Escherichia coli	en_US
dc.subject.mesh	Dihydroxyphenylalanine	en_US
dc.subject.mesh	Peptidylprolyl Isomerase	en_US
dc.subject.mesh	Cyclophilins	en_US
dc.subject.mesh	Tyrosine	en_US
dc.subject.mesh	Histidine	en_US
dc.subject.mesh	Peptides	en_US
dc.subject.mesh	Proteins	en_US
dc.subject.mesh	Chromatography, High Pressure Liquid	en_US
dc.subject.mesh	Magnetic Resonance Spectroscopy	en_US
dc.subject.mesh	Spectrometry, Mass, Electrospray Ionization	en_US
dc.subject.mesh	Protein Engineering	en_US
dc.subject.mesh	Protein Biosynthesis	en_US
dc.subject.mesh	Amino Acid Sequence	en_US
dc.subject.mesh	Molecular Sequence Data	en_US
dc.title	Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins	en_US
dc.type	Journal Article
utslib.citation.volume	12	en_US
utslib.citation.volume	272	en_US
utslib.for	0601 Biochemistry and Cell Biology	en_US
utslib.for	0304 Medicinal and Biomolecular Chemistry	en_US
utslib.for	1101 Medical Biochemistry and Metabolomics	en_US
dc.location.activity	ISI:000230088000023	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Faculty of Science/School of Life Sciences
pubs.organisational-group	/University of Technology Sydney/Strength - CHT - Health Technologies
utslib.copyright.status	closed_access
pubs.issue	12	en_US
pubs.publication-status	Published	en_US
pubs.volume	272	en_US

Abstract:

An Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation of L-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 clean 15N-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. © 2005 FEBS.

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http://hdl.handle.net/10453/14959