Vol. 3, No. 2 Products For Biomolecular NMR Download PDF version (1.49 MB)

Volume 3 Number 2 Products for Biomolecular NMR Modern multiple resonance and multi-dimensonal Biomolecular NMR experiments require isotopic enrichment of the proteins, RNA and DNA, to achieve sufficient sensitivity and resolution. The biomolecules are typically enriched by biosynthesis. ISOTEC™ offers an extensive line of stable isotope labeled products for Biomolecular NMR applications.

Stable Isotopes in Cell-Free Protein Synthesis

Munehiro Teshima-Scientist-ISOTEC™

Proteomics is an emerging field of post genome research, which examines the enormous amounts of information stored in the genomes of living organisms.¹ Biomolecular NMR and stable isotopes are important tools used to study the structure and function of proteins in structural genomics research. Most researchers use the protein expression systems of microorganisms or mammalian cells and stable isotope labeled (²H, ¹³C, ¹⁵N) D-glucose, ammonium salts, amino acids and/or complex growth media to produce proteins uniformly labeled with stable isotopes.

However, the protein expression system using living cells has limitations.

• Many expressed proteins are insoluble and aggregate in inclusion bodies.
• Intercellular proteases in the host cells may digest the proteins.
• Some proteins cannot be produced in living cells because of their toxicity.
• The amino acid metabolic system in the host cells can cause isotope dilution and diffusion for amino acids
• with selective stable isotope labeling.
• It may be difficult to grow organisms in deuterium labeled media.
• Procaryote cannot glycosylate proteins.

These problems associated with living cells may be solved by the cell-free protein synthesis system. An additional benefit is that the cell-free protein synthesis system is suitable for automation and high throughput protein synthesis. The basic idea of the cell-free protein synthesis system is to extract only the protein synthesis system (translation and / or transcription system) from living cells such as Escherichia Coli, rabbit reticulocyte or wheat germ, and allow the extract to synthesize the proteins in vitro from amino acids and specific DNA or mRNA used as a template. The cell-free protein synthesis system is not a new technology, but the yield of the conventional batch type system used in the past was so low that radioisotopes were the only method sensitive enough for detection. In 1988, Dr. Spirin et al. developed a continuous flow cell-free protein synthesis system. They used an ultra-filtration system and succeeded in producing a couple of hundred mg of protein per ml of reaction mixture.2

Many ideas for the improvement of the continuous flow system such as using dialysis and condensation of the extract were incorporated by many researchers.^3-7 In addition to these improvements, Dr. Yokoyama et al. optimized the reaction conditions and established a system to produce 6 mg of protein per ml of reaction mixture, and they applied this system to produce ¹³C, ¹⁵N-labeled proteins for Biomolecular NMR spectroscopy.⁸ Also, they succeeded in developing a site directed stable isotope labeling method for a protein by using a cell-free protein synthesis system.⁹

References:

1. http://www.gsc.riken.go.jp/e/group/protgrE.html.
2. Sprin, A. S., et al., Science, 1988, 1162-1164, 242.
3. Kim, D. M., et al., Biotechnol. Prog., 1996, 645-649, 12.
4. Davis, J., et al., Promega Notes Mag. 1996, 14-18, 56.
5. Nakano, H., et al., Biosci. Biotechnol. Biochem. 1994, 631-634, 58.
6. Kim, D. M., et al., Eur. J. Biochem. 1996, 881-886, 239.
7. Nakano, H., et al., J. Biotechnol. 1996, 275-282, 46.
8. Yokoyama, S., et al., FEBS Lett. 1999, 15-19, 442.
9. Yokoyama, S., et al., J. Biomol. NMR 1998, 295-306, 11.

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