Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O
Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O
Abstract Selectively isotope labelled protein samples can be prepared in vivo or in vitro from selectively labelled amino acids but, in many cases, metabolic conversions between different amino acids result in isotope scrambling. The best results are obtained by cell-free protein synthesis, where metabolic enzymes are generally less active, but isotope scrambling can never be suppressed completely. We show that reduction of E. coli S30 extracts with NaBH4 presents a simple and inexpensive way to achieve cleaner selective isotope labelling in cell-free protein synthesis reactions. The purpose of the NaBH4 is to inactivate all pyridoxal-phosphate (PLP) dependent enzymes by irreversible reduction of the Schiff bases formed between PLP and lysine side chains of the enzymes or amino groups of free amino acids. The reduced S30 extracts retain their activity of protein synthesis, can be stored as well as conventional S30 extracts and effectively suppress conversions between different amino acids. In addition, inactivation of PLP-dependent enzymes greatly stabilizes hydrogens bound to α-carbons against exchange with water, minimizing the loss of α-deuterons during cell-free production of proteins from perdeuterated amino acids in H2O solution. This allows the production of highly perdeuterated proteins that contain protons at all exchangeable positions, without having to back-exchange labile deuterons for protons as required for proteins that have been synthesized in D2O.
Content Type Journal Article
Pages 1-8
DOI 10.1007/s10858-011-9477-5
Authors
Xun-Cheng Su, Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia
Choy-Theng Loh, Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia
Ruhu Qi, Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia
Gottfried Otting, Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia
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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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Cell-free protein synthesis of perdeuterated proteins for NMR studies
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Touraj Etezady-Esfarjani, Sebastian Hiller, Cristina Villalba and Kurt Wüthrich
Journal of Biomolecular NMR; 2007; 39(3); pp 229-238
Abstract:
Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels. This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen atom positions. Potential applications of the new protocol may include production of proteins...
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Wheat Germ Cell-Free Protein Production Workshop
Wheat Germ Cell-Free Protein Production Workshop
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The Center for Eukaryotic Structural Genomics (CESG) and the Nuclear Magnetic Resonance Facility at Madison
(NMRFAM) are pleased to announce the first Wheat Germ Cell-Free Protein Production Workshop to be held from
July 30 - August 4, 2006, at the Department of Biochemistry at the University of Wisconsin-Madison in Madison, Wisconsin,
USA. To register for this workshop, complete the Registration Form on the next page and mail in with your NONREFUNDABLE
Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O
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