Abstract Using the single-protein-production (SPP) system, a protein of interest can be exclusively produced in high yield from its ACA-less gene in Escherichia coli expressing MazF, an ACA-specific mRNA interferase. It is thus feasible to study a membrane protein by solid-state NMR (SSNMR) directly in natural membrane fractions. In developing isotope-enrichment methods, we observed that 13C was also incorporated into phospholipids, generating spurious signals in SSNMR spectra. Notable, with the SPP system a protein can be produced in total absence of cell growth caused by antibiotics. Here, we demonstrate that cerulenin, an inhibitor of phospholipid biosynthesis, can suppress isotope incorporation in the lipids without affecting membrane protein yield in the SPP system. SSNMR analysis of ATP synthase subunit c, an E. coli inner membrane protein, produced by the SPP method using cerulenin revealed that 13C resonance signals from phospholipid were markedly reduced, while signals for the isotope-enriched protein were clearly present.
Content Type Journal Article
Pages 1-7
DOI 10.1007/s10858-011-9469-5
Authors
Lili Mao, Department of Biochemistry, Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA
Koichi Inoue, Department of Biochemistry, Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA
Yisong Tao, Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA
Gaetano T. Montelione, Department of Biochemistry, Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA
Ann E. McDermott, Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA
Masayori Inouye, Department of Biochemistry, Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA
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...
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[NMR paper] Cell-free protein synthesis in an autoinduction system for NMR studies of protein-protein interactions.
Cell-free protein synthesis in an autoinduction system for NMR studies of protein-protein interactions.
Related Articles Cell-free protein synthesis in an autoinduction system for NMR studies of protein-protein interactions.
J Biomol NMR. 2005 Jul;32(3):235-41
Authors: Ozawa K, Jergic S, Crowther JA, Thompson PR, Wijffels G, Otting G, Dixon NA
Cell-free protein synthesis systems provide facile access to proteins in a nascent state that enables formation of soluble, native protein-protein complexes even if one of the protein components is prone...
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12-01-2010 06:56 PM
[NMR paper] Biosynthesis and NMR analysis of a 73-residue domain of a Saccharomyces cerevisiae G protein-coupled receptor.
Biosynthesis and NMR analysis of a 73-residue domain of a Saccharomyces cerevisiae G protein-coupled receptor.
Related Articles Biosynthesis and NMR analysis of a 73-residue domain of a Saccharomyces cerevisiae G protein-coupled receptor.
Biochemistry. 2005 Sep 6;44(35):11795-810
Authors: Estephan R, Englander J, Arshava B, Samples KL, Becker JM, Naider F
The yeast Saccharomyces cerevisiae alpha-factor pheromone receptor (Ste2p) was used as a model G protein-coupled receptor (GPCR). A 73-mer multidomain fragment of Ste2p (residues 267-339)...
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[NMR paper] Expanding the Scope of Protein Biosynthesis by Altering the Methionyl-tRNA Synthetase
Expanding the Scope of Protein Biosynthesis by Altering the Methionyl-tRNA Synthetase Activity of a Bacterial Expression Host Scott Ross was helpful in conducting the 1D TOCSY NMR experiments and Pratip Bhattachary is thanked for assistance in other NMR experiments. We are grateful to Yves Mechulam for a sample of plasmid pBSM547W305F and to Hieronim Jakubowski of UMDNJ-New Jersey Medical School, Newark, New Jersey, for plasmid pGG3. K.L.K. thanks the U.S. Department of Defense for a National Defense Science and Engineering Graduate Fellowship. This work was supported by grants from the...
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Efficient protein production method for NMR using soluble protein tags with cold shoc
Efficient protein production method for NMR using soluble protein tags with cold shock expression vector
Abstract The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins...
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09-18-2010 04:53 AM
Efficient protein production method for NMR using soluble protein tags with cold shoc
Efficient protein production method for NMR using soluble protein tags with cold shock expression vector.
Related Articles Efficient protein production method for NMR using soluble protein tags with cold shock expression vector.
J Biomol NMR. 2010 Sep 16;
Authors: Hayashi K, Kojima C
The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To...
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09-17-2010 04:14 PM
Breakthrough in protein production for NMR?
The next best thing after cryoprobe and TROSY?
Single Protein Production in Living Cells Facilitated by an mRNA Interferase
Motoo Suzuki,1 Junjie Zhang,1 Mohan Liu,2 Nancy A. Woychik,2 and Masayori Inouye1,*
1 Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854
2 Department of Molecular Genetics, Microbiology and Immunology, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854
Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system
Linear Mode
