Related ArticlesDictyostelium discoideum as expression host: isotopic labeling of a recombinant glycoprotein for NMR studies.
Protein Expr Purif. 2000 Aug;19(3):335-42
Authors: Cubeddu L, Moss CX, Swarbrick JD, Gooley AA, Williams KL, Curmi PM, Slade MB, Mabbutt BC
The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [(15)N]NH(4)Cl and [(13)C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly (13)C,(15)N-labeled protein secreted by approximately 10(10) D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional (1)H-(13)C HSQC spectrum confirms (13)C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.
31P NMR correlation maps of 18O/16O chemical shift isotopic effects for phosphometabolite labeling studies
31P NMR correlation maps of 18O/16O chemical shift isotopic effects for phosphometabolite labeling studies
Abstract Intramolecular correlations among the 18O-labels of metabolic oligophosphates, mapped by J-decoupled 31P NMR 2D chemical shift correlation spectroscopy, impart stringent constraints to the 18O-isotope distributions over the whole oligophosphate moiety. The multiple deduced correlations of isotopic labels enable determination of site-specific fractional isotope enrichments and unravel the isotopologue statistics. This approach ensures accurate determination of 18O-labeling...
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[NMR paper] Segmental isotopic labeling for structural biological applications of NMR.
Segmental isotopic labeling for structural biological applications of NMR.
Related Articles Segmental isotopic labeling for structural biological applications of NMR.
Methods Mol Biol. 2004;278:47-56
Authors: Cowburn D, Shekhtman A, Xu R, Ottesen JJ, Muir TW
This chapter describes the preparation of precursor domains for the formation of multidomain segmentally labeled proteins by protein ligation.
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[NMR paper] Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and ir
Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.
Related Articles Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.
J Biomol NMR. 2000 Aug;17(4):311-22
Authors: Sorkin DL, Miller AF
We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site 1H NMR resonances of iron(II)- and iron(III)-superoxide...
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11-19-2010 08:29 PM
[NMR paper] Chain-selective isotopic labeling for NMR studies of large multimeric proteins: appli
Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.
Related Articles Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.
Biophys J. 2000 Aug;79(2):1146-54
Authors: Simplaceanu V, Lukin JA, Fang TY, Zou M, Ho NT, Ho C
Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of...
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[NMR paper] Chemical ligation of folded recombinant proteins: segmental isotopic labeling of doma
Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies.
Related Articles Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies.
Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):388-93
Authors: Xu R, Ayers B, Cowburn D, Muir TW
A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology...
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11-18-2010 07:05 PM
[NMR paper] Labeling of recombinant protein for NMR spectroscopy: global and specific labeling of
Labeling of recombinant protein for NMR spectroscopy: global and specific labeling of the rat liver fructose 2,6-bisphosphatase domain.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Labeling of recombinant protein for NMR spectroscopy: global and specific labeling of the rat liver fructose 2,6-bisphosphatase domain.
Protein Expr Purif. 1997 Oct;11(1):79-85
Authors: Okar DA, Felicia ND, Gui L, Lange AJ
Methods for the efficient use of the 13C-labeled nutrients,...
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[NMR paper] Protein expression, selective isotopic labeling, and analysis of hyperfine-shifted NM
Protein expression, selective isotopic labeling, and analysis of hyperfine-shifted NMR signals of Anabaena 7120 vegetative ferredoxin.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Protein expression, selective isotopic labeling, and analysis of hyperfine-shifted NMR signals of Anabaena 7120 vegetative ferredoxin.
Arch Biochem Biophys. 1995 Jan 10;316(1):619-34
Authors: Cheng H, Westler WM, Xia B, Oh BH, Markley JL
Two alternative T7 RNA promoter/polymerase systems...
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[NMR paper] Heteronuclear 3D NMR and isotopic labeling of calmodulin. Towards the complete assign
Heteronuclear 3D NMR and isotopic labeling of calmodulin. Towards the complete assignment of the 1H NMR spectrum.
Related Articles Heteronuclear 3D NMR and isotopic labeling of calmodulin. Towards the complete assignment of the 1H NMR spectrum.
Biochem Pharmacol. 1990 Jul 1;40(1):153-60
Authors: Ikura M, Marion D, Kay LE, Shih H, Krinks M, Klee CB, Bax A
New methods are described that permit detailed analysis of the NMR spectra of calmodulin, an alpha-helical protein with a molecular weight of 16.7 kD. Two complementary approaches have been...
Dictyostelium discoideum as expression host: isotopic labeling of a recombinant glyco
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