caa a22 4500 445325666 CHVBK 20180317142719.0 cr unu---uuuuu 170323e20110501xx s 000 0 eng 10.1007/s10858-011-9477-5 doi (NATIONALLICENCE)springer-10.1007/s10858-011-9477-5 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 [Elektronische Daten] [Xun-Cheng Su, Choy-Theng Loh, Ruhu Qi, Gottfried Otting] 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. Springer Science+Business Media B.V., 2011 Cell-free protein synthesis nationallicence Isotope scrambling nationallicence NaBH4 nationallicence Pyridoxal phosphate nationallicence Selective 15N labelling nationallicence Su Xun-Cheng Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut Loh Choy-Theng Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut Qi Ruhu Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut Otting Gottfried Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut Journal of Biomolecular NMR Springer Netherlands 50/1(2011-05-01), 35-42 0925-2738 50:1<35 2011 50 10858 https://doi.org/10.1007/s10858-011-9477-5 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-011-9477-5 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Su Xun-Cheng Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut NATIONALLICENCE 700 1- Loh Choy-Theng Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut NATIONALLICENCE 700 1- Qi Ruhu Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut NATIONALLICENCE 700 1- Otting Gottfried Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 50/1(2011-05-01), 35-42 0925-2738 50:1<35 2011 50 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer