Related ArticlesProtocol for aerosol-free recombinant production and NMR analysis of prion proteins.
J Biomol NMR. 2014 Apr 26;
Authors: Rehbein P, Saxena K, Schlepckow K, Schwalbe H
Abstract
The central hallmark of prion diseases is the misfolding of cellular prion protein (PrP(C)) into a disease-associated aggregated isoform known as scrapie prion protein (PrP(Sc)). NMR spectroscopy has made many essential contributions to the characterization of recombinant PrP in its folded, unfolded and aggregated states. Recent studies reporting on de novo generation of prions from recombinant PrP and infection of animals using prion aerosols suggest that adjustment of current biosafety measures may be necessary, particularly given the relatively high protein concentrations required for NMR applications that favor aggregation. We here present a protocol for the production of recombinant PrP under biosafety level 2 conditions that avoids entirely exposure of the experimenter to aerosols that might contain harmful PrP aggregates. In addition, we introduce an NMR sample tube setup that allows for safe handling of PrP samples at the spectrometer that usually is not part of a dedicated biosafety level 2 laboratory.
PMID: 24771297 [PubMed - as supplied by publisher]
[NMR paper] The production of recombinant (15)N, (13)C-labelled somatostatin 14 for NMR spectroscopy.
The production of recombinant (15)N, (13)C-labelled somatostatin 14 for NMR spectroscopy.
The production of recombinant (15)N, (13)C-labelled somatostatin 14 for NMR spectroscopy.
Protein Expr Purif. 2014 Mar 31;
Authors: Nespovitaya N, Barylyuk K, Eichmann C, Zenobi R, Riek R
Abstract
Structural studies of human peptide hormone somatostatin 14 (SS14) require high amounts of isotopically labelled SS14 to be produced. Here we report a method for effective production of isotopically labelled SS14. SS14 was expressed as a fusion...
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[NMR paper] A Cost-Effective Protocol for the Parallel Production of Libraries of (13)CH 3-Specifically Labeled Mutants for NMR Studies of High Molecular Weight Proteins.
A Cost-Effective Protocol for the Parallel Production of Libraries of (13)CH 3-Specifically Labeled Mutants for NMR Studies of High Molecular Weight Proteins.
Related Articles A Cost-Effective Protocol for the Parallel Production of Libraries of (13)CH 3-Specifically Labeled Mutants for NMR Studies of High Molecular Weight Proteins.
Methods Mol Biol. 2014;1091:229-44
Authors: Crublet E, Kerfah R, Mas G, Noirclerc-Savoye M, Lantez V, Vernet T, Boisbouvier J
Abstract
There is increasing interest in applying NMR spectroscopy to the study of...
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11-11-2013 01:30 AM
An efficient protocol for incorporation of an unnatural amino acid in perdeuterated recombinant proteins using glucose-based media
An efficient protocol for incorporation of an unnatural amino acid in perdeuterated recombinant proteins using glucose-based media
Abstract The in vivo incorporation of unnatural amino acids into proteins is a well-established technique requiring an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is incorporated at a position encoded by a TAG amber codon. Although this technology provides unique opportunities to engineer protein structures, poor protein yields are usually obtained in deuterated media, hampering its application in the protein NMR...
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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02-16-2011 09:34 PM
[NMR paper] Cell-free protein production and labeling protocol for NMR-based structural proteomic
Cell-free protein production and labeling protocol for NMR-based structural proteomics.
Related Articles Cell-free protein production and labeling protocol for NMR-based structural proteomics.
Nat Methods. 2004 Nov;1(2):149-53
Authors: Vinarov DA, Lytle BL, Peterson FC, Tyler EM, Volkman BF, Markley JL
Structural proteomics requires robust, scalable methods. Here we describe a wheat germ cell-free platform for protein production that supports efficient NMR structural studies of eukaryotic proteins and offers advantages over cell-based methods....
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11-24-2010 10:01 PM
[NMR paper] Recombinant production of the p10CKS1At protein from Arabidopsis thaliana and 13C and
Recombinant production of the p10CKS1At protein from Arabidopsis thaliana and 13C and 15N double-isotopic enrichment for NMR studies.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Recombinant production of the p10CKS1At protein from Arabidopsis thaliana and 13C and 15N double-isotopic enrichment for NMR studies.
Protein Expr Purif. 1999 Jun;16(1):144-51
Authors: Landrieu I, Casteels P, Odaert B, De Veylder L, Portetelle D, Lippens G, Van Montagu M, Inzé D
The CKS1At...
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08-21-2010 04:03 PM
Enhanced production and isotope enrichment of recombinant glycoproteins produced in c
Abstract NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant...
Protocol for aerosol-free recombinant production and NMR analysis of prion proteins.
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