Journal of Biomolecular NMR

, Volume 53, Issue 1, pp 43–51 | Cite as

High-yield Escherichia coli-based cell-free expression of human proteins

  • Erich Michel
  • Kurt Wüthrich


Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease activity. In the implementation of the expression system we used freshly in-house prepared cell extract. Batch-mode cell-free expression with this setup was up to twofold more economical than continuous-exchange expression, with yields of 0.2–0.9 mg of purified protein per mL of reaction mixture. Native folding of the proteins thus obtained is documented with 2D [15N,1H]-HSQC NMR.


Batch-mode cell-free protein expression Escherichia coli S30 cell extract Stable-isotope labeling Structural biology of human proteins 





Batch mode cell-free


Base pairs


Continuous-exchange cell-free






Dihydrofolate reductase




Ethylenediaminetetraacetic acid


Adipocyte fatty acid-binding protein 4


Human peptidyl-prolyl cis–trans isomerase FKBP1A


B1 domain of protein G from Streptococcus sp


γ-Interferon-inducible lysosomal thiol reductase


Heteronuclear single quantum coherence




Luria Bertani


Mitochondrial methylmalonyl-CoA epimerase


Methenyl-THF synthetase




Phosphate-buffered saline


Purkinje cell protein 2 homolog


Peptidyl-prolyl cis–trans isomerase B


Phosphate/yeast extract/glucose


Sodium dodecyl-sulfate polyacrylamide gel electrophoresis


Stromal cell-derived growth factor


RNA polymerase from bacteriophage T7


Tris-buffered saline



We thank Cristina Stocker for help with cell extract preparations, Dr. Arthur Horwich for providing the pET21-DHFR plasmid, Dr. Eilika Weber-Ban for providing the pET19-TEV plasmid, and the Swiss National Science Foundation and the ETH Zürich for financial support through the National Center of Competence in Research (NCCR) Structural Biology.

Supplementary material

10858_2012_9619_MOESM1_ESM.docx (28 kb)
Supplementary material 1 (DOCX 29 kb)
10858_2012_9619_MOESM2_ESM.tif (5.4 mb)
Supplementary material 2 (TIFF 5567 kb)


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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  1. 1.Institute of Molecular Biology and BiophysicsETH ZurichZurichSwitzerland

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