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
- 284 Downloads
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.
KeywordsCell-free protein synthesis Isotope scrambling NaBH4 Pyridoxal phosphate Selective 15N labelling
We thank Mr. Xinying Jia and Dr. Hiromasa Yagi for help with the NMR spectroscopy and Cambridge Isotope Laboratories for generous gifts of 2H/15N/13C-labelled amino acids and amino acid mixture. Financial support by the Australian Research Council is gratefully acknowledged.
- Apponyi MA, Ozawa K, Dixon NE, Otting G (2008) Cell-free protein synthesis for analysis by NMR spectroscopy. In: Kobe B, Guss M, Huber T (eds) Methods in molecular biology 426, structural proteomics: high-throughput methods. Humana Press, Totowa, pp 257–268Google Scholar
- Berg JM, Tymoczko JL, Stryer L (2006) Biochemistry. WH Freeman and Company, New YorkGoogle Scholar
- Michuda CM, Martinez-Carrion M (1970) The isozymes of glutamate-aspartate transaminase. Mechanism of inhibition by dicarboxylic acids. J Biol Chem 245:262–269Google Scholar
- Prusiner S, Stadtman ER (1976) Regulation of glutaminase B in Escherichia coli. II. Modulation of activity by carboxylate and borate ions. J Biol Chem 251:3457–3462Google Scholar
- Weisbrod RE, Meister A (1973) Studies on glutamine synthetase from Escherichia coli: formation of pyrrolidone carboxylate and inhibition by methionine sulfoximine. J Biol Chem 248:3997–4002Google Scholar