Details
| Original language | English |
|---|---|
| Pages (from-to) | 823-833 |
| Number of pages | 11 |
| Journal | Applied Microbiology and Biotechnology |
| Volume | 92 |
| Issue number | 4 |
| Early online date | 9 Oct 2011 |
| Publication status | Published - Nov 2011 |
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 ( 15N, 13C, and 2H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for 15N and 13C, respectively, and 75% of (non-exchangeable) hydrogen for 2H 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.
Keywords
- Autoinduction, Defined medium, Escherichia coli, Recombinant protein production, Selenomethionine labeling, Stable heavy isotope labeling
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
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In: Applied Microbiology and Biotechnology, Vol. 92, No. 4, 11.2011, p. 823-833.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems
AU - Li, Zhaopeng
AU - Nimtz, Manfred
AU - Rinas, Ursula
PY - 2011/11
Y1 - 2011/11
N2 - 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 ( 15N, 13C, and 2H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for 15N and 13C, respectively, and 75% of (non-exchangeable) hydrogen for 2H 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.
AB - 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 ( 15N, 13C, and 2H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for 15N and 13C, respectively, and 75% of (non-exchangeable) hydrogen for 2H 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.
KW - Autoinduction
KW - Defined medium
KW - Escherichia coli
KW - Recombinant protein production
KW - Selenomethionine labeling
KW - Stable heavy isotope labeling
UR - http://www.scopus.com/inward/record.url?scp=82355181066&partnerID=8YFLogxK
U2 - 10.1007/s00253-011-3603-x
DO - 10.1007/s00253-011-3603-x
M3 - Article
C2 - 21983707
AN - SCOPUS:82355181066
VL - 92
SP - 823
EP - 833
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
SN - 0175-7598
IS - 4
ER -