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

Xun Cheng Su, Choy Theng Loh, Ruhu Qi, Gottfried Otting*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    34 Citations (Scopus)

    Abstract

    Selectively isotope labelled protein samples can be prepared in vivo or in vitro from selectively labeled 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 NaBH 4 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.

    Original languageEnglish
    Pages (from-to)35-42
    Number of pages8
    JournalJournal of Biomolecular NMR
    Volume50
    Issue number1
    DOIs
    Publication statusPublished - May 2011

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