Related ArticlesHigh-throughput construction method for expression vector of peptides for NMR study suited for isotopic labeling.
Protein Eng Des Sel. 2004 Apr;17(4):305-14
Authors: Tenno T, Goda N, Tateishi Y, Tochio H, Mishima M, Hayashi H, Shirakawa M, Hiroaki H
Fusion protein constructs for labeled peptides were generated with the 114 amino acid thioredoxin (TRX), coupled with the incorporation of a histidine tag for affinity purification. Two tandem AhdI sites were designed in the multiple cloning site of the fusion vector according to our novel unidirectional TA cloning methodology named PRESAT-vector, allowing one-step background-free cloning of DNA fragments. Constructs were designed to incorporate the four residue sequence Ile-Asp-Gly-Arg to generate pure peptides following Factor Xa cleavage of the fusion protein. The system is efficient and cost-effective for isotopic labeling of peptides for heteronuclear NMR studies. Seven peptides of varying length, including pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and ubiquitin interacting motif (UIM), were expressed using this TRX fusion system to give soluble fusion protein constructs in all cases. Three alternative methods for the preparation of DNA fragments were applied depending on the length of the peptides, such as polymerase chain reaction, chemical synthesis or a 'semi-synthetic method', which is a combination of chemical synthesis and enzymatic extension. The ability easily to construct, express and purify recombinant peptides in a high-throughput manner will be of enormous benefit in areas of biomedical research and drug discovery.
[NMR paper] High-throughput 3D structural homology detection via NMR resonance assignment.
High-throughput 3D structural homology detection via NMR resonance assignment.
Related Articles High-throughput 3D structural homology detection via NMR resonance assignment.
Proc IEEE Comput Syst Bioinform Conf. 2004;:278-89
Authors: Langmead CJ, Donald BR
One goal of the structural genomics initiative is the identification of new protein folds. Sequence-based structural homology prediction methods are an important means for prioritizing unknown proteins for structure determination. However, an important challenge remains: two highly...
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11-24-2010 09:25 PM
[NMR paper] High-throughput screening of structural proteomics targets using NMR.
High-throughput screening of structural proteomics targets using NMR.
Related Articles High-throughput screening of structural proteomics targets using NMR.
FEBS Lett. 2003 Sep 25;552(2-3):207-13
Authors: Galvão-Botton LM, Katsuyama AM, Guzzo CR, Almeida FC, Farah CS, Valente AP
We applied a high-throughput strategy for the screening of targets for structural proteomics of Xanthomonas axonopodis pv citri. This strategy is based on the rapid (1)H-(15)N HSQC NMR analysis of bacterial lysates containing selectively (15)N-labelled heterologous...
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11-24-2010 09:16 PM
[NMR paper] Fluorine-NMR experiments for high-throughput screening: theoretical aspects, practica
Fluorine-NMR experiments for high-throughput screening: theoretical aspects, practical considerations, and range of applicability.
Related Articles Fluorine-NMR experiments for high-throughput screening: theoretical aspects, practical considerations, and range of applicability.
J Am Chem Soc. 2003 Jun 25;125(25):7696-703
Authors: Dalvit C, Fagerness PE, Hadden DT, Sarver RW, Stockman BJ
Competition ligand-based NMR screening experiments have recently been introduced to overcome most of the problems associated with traditional ligand-based NMR...
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11-24-2010 09:01 PM
[NMR paper] Integration of NMR and high-throughput screening.
Integration of NMR and high-throughput screening.
Related Articles Integration of NMR and high-throughput screening.
Comb Chem High Throughput Screen. 2002 Dec;5(8):613-21
Authors: Hajduk PJ, Burns DJ
NMR-based screening has become a powerful method for the identification and analysis of low-molecular weight organic compounds that bind to protein targets and can be utilized in drug discovery programs. In particular, heteronuclear NMR-based screening can yield information about both the affinity and binding location of potential lead compounds....
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[NMR paper] NMR analysis of in vitro-synthesized proteins without purification: a high-throughput
NMR analysis of in vitro-synthesized proteins without purification: a high-throughput approach.
Related Articles NMR analysis of in vitro-synthesized proteins without purification: a high-throughput approach.
FEBS Lett. 2002 Jul 31;524(1-3):159-62
Authors: Guignard L, Ozawa K, Pursglove SE, Otting G, Dixon NE
A cell-free protein expression system was established that provides protein samples of adequate concentration and purity for direct NMR analysis. The Escherichia coli peptidyl-prolyl cis-trans isomerase PpiB was expressed in this system...
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[NMR paper] Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignm
Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignment.
Related Articles Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignment.
Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):8009-14
Authors: Szyperski T, Yeh DC, Sukumaran DK, Moseley HN, Montelione GT
A suite of reduced-dimensionality (13)C,(15)N,(1)H-triple-resonance NMR experiments is presented for rapid and complete protein resonance assignment. Even when using short measurement times, these experiments allow one to retain...
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11-24-2010 08:49 PM
[NMR paper] An approach for high-throughput structure determination of proteins by NMR spectrosco
An approach for high-throughput structure determination of proteins by NMR spectroscopy.
Related Articles An approach for high-throughput structure determination of proteins by NMR spectroscopy.
J Biomol NMR. 2000 Nov;18(3):229-38
Authors: Medek A, Olejniczak ET, Meadows RP, Fesik SW
An approach is described for rapidly determining protein structures by NMR that utilizes proteins containing 13C-methyl labeled Val, Leu, and Ile (delta1) and protonated Phe and Tyr in a deuterated background. Using this strategy, the key NOEs that define the...
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11-19-2010 08:29 PM
NMR in a crystallography-based high-throughput protein structure-determination enviro
NMR in a crystallography-based high-throughput protein structure-determination environment.
Related Articles NMR in a crystallography-based high-throughput protein structure-determination environment.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Oct 1;66(Pt 10):1365-6
Authors: Wüthrich K
An introduction is provided to three papers which compare corresponding protein crystal and NMR solution structures determined by the Joint Center for Structural Genomics (JCSG). Special mention is made of the JCSG strategy for combined use of the two...