Abstract 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 0.2â??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.
Content Type Journal Article
Category Article
Pages 1-9
DOI 10.1007/s10858-012-9619-4
Authors
Erich Michel, Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland
Kurt Wüthrich, Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland
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Protein science is shifting towards experiments performed under native or native-like conditions. In-cell NMR spectroscopy for instance has the potential to reveal protein structure and dynamics inside cells. However, not all proteins can be studied by this technique. (15) N-labelled...
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Cell-free expression and stable isotope labelling strategies for membrane proteins
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Abstract Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems...
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SH3 domain binding glutamic acid-rich protein like 3 (SH3BGRL3) is the new member of thioredoxin (TRX) super family, whose posttranslational modified form was identified as tumor necrosis factor alpha (TNF-alpha) inhibitory protein, TIP-B1. In this paper,...
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We have developed an efficient and novel filter assay method, involving radioactive labelling and imaging, to quantify the expression of soluble proteins from a...
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Structural proteomics requires robust, scalable methods. Here we describe a wheat germ cell-free platform for protein production that supports efficient NMR structural studies of eukaryotic proteins and offers advantages over cell-based methods....
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Cell-free protein synthesis offers rapid access to proteins that are selectively labelled with amino acids and suitable for...
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Authors: Berglechner F, Richter G, Fischer M, Bacher A, Gschwind RM,...
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N Biotechnol. 2010 Aug 3;
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Although solid-state NMR and cell-free expression have recently become standard methods in biology, the combination of the two is still at a very early stage of development. In this...
High-yield Escherichia coli-based cell-free expression of human proteins
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