Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems.

2.50
Hdl Handle:
http://hdl.handle.net/10033/221512
Title:
Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems.
Authors:
Li, Zhaopeng; Nimtz, Manfred; Rinas, Ursula
Abstract:
Generating sufficient quantities of labeled proteins represents a bottleneck in protein structure determination. A simple protocol for producing heavy isotope as well as selenomethionine (Se-Met)-labeled proteins was developed using T7-based Escherichia coli expression systems. The protocol is applicable for generation of single-, double-, and triple-labeled proteins ((15)N, (13)C, and (2)H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for (15)N and (13)C, respectively, and 75% of (non-exchangeable) hydrogen for (2)H labeling. The expression yields and final cell densities (OD600 ~16) were the same as for the production of non-labeled protein. This protocol is also applicable for Se-Met labeling, leading to Se-Met incorporation into the target protein of 70% or 90% using prototrophic or methionine auxotrophic E. coli strains, respectively.
Affiliation:
Helmholtz Centre for Infection Research (SB), Braunschweig, Germany.
Citation:
Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems. 2011, 92 (4):823-33 Appl. Microbiol. Biotechnol.
Journal:
Applied microbiology and biotechnology
Issue Date:
Nov-2011
URI:
http://hdl.handle.net/10033/221512
DOI:
10.1007/s00253-011-3603-x
PubMed ID:
21983707
Type:
Article
Language:
en
ISSN:
1432-0614
Appears in Collections:
Publications of the RG Rekombinante Proteinexpression (RPEX)

Full metadata record

DC FieldValue Language
dc.contributor.authorLi, Zhaopengen_GB
dc.contributor.authorNimtz, Manfreden_GB
dc.contributor.authorRinas, Ursulaen_GB
dc.date.accessioned2012-05-02T09:54:29Z-
dc.date.available2012-05-02T09:54:29Z-
dc.date.issued2011-11-
dc.identifier.citationOptimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems. 2011, 92 (4):823-33 Appl. Microbiol. Biotechnol.en_GB
dc.identifier.issn1432-0614-
dc.identifier.pmid21983707-
dc.identifier.doi10.1007/s00253-011-3603-x-
dc.identifier.urihttp://hdl.handle.net/10033/221512-
dc.description.abstractGenerating sufficient quantities of labeled proteins represents a bottleneck in protein structure determination. A simple protocol for producing heavy isotope as well as selenomethionine (Se-Met)-labeled proteins was developed using T7-based Escherichia coli expression systems. The protocol is applicable for generation of single-, double-, and triple-labeled proteins ((15)N, (13)C, and (2)H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for (15)N and (13)C, respectively, and 75% of (non-exchangeable) hydrogen for (2)H labeling. The expression yields and final cell densities (OD600 ~16) were the same as for the production of non-labeled protein. This protocol is also applicable for Se-Met labeling, leading to Se-Met incorporation into the target protein of 70% or 90% using prototrophic or methionine auxotrophic E. coli strains, respectively.en_GB
dc.language.isoenen
dc.rightsArchived with thanks to Applied microbiology and biotechnologyen_GB
dc.subject.meshCarbon Isotopesen_GB
dc.subject.meshDeuteriumen_GB
dc.subject.meshEscherichia colien_GB
dc.subject.meshGene Expressionen_GB
dc.subject.meshGenetic Vectorsen_GB
dc.subject.meshIsotope Labelingen_GB
dc.subject.meshNitrogen Isotopesen_GB
dc.subject.meshProteinsen_GB
dc.subject.meshSelenomethionineen_GB
dc.titleOptimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems.en
dc.typeArticleen
dc.contributor.departmentHelmholtz Centre for Infection Research (SB), Braunschweig, Germany.en_GB
dc.identifier.journalApplied microbiology and biotechnologyen_GB

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