title: Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems.
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.
authors: Helmholtz Centre for Infection Research (SB), Braunschweig, Germany.
authors: Zhaopeng
authors: Z
authors: Li
authors: Manfred
authors: M
authors: Nimtz
authors: Ursula
authors: U
authors: Rinas
mesh_terms: D002247:Carbon Isotopes / Q000378:metabolism*; D003903:Deuterium / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012645:Selenomethionine / Q000378:metabolism*
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D012645:Selenomethionine; D003903:Deuterium