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NMR processing:
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Side-chains:
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NOEs:
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UNIO Candid
ASDP
Structure from NMR restraints:
Ab initio:
GeNMR
Cyana
XPLOR-NIH
ASDP
UNIO ATNOS-Candid
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Fragment-based:
BMRB CS-Rosetta
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Template-based:
GeNMR
I-TASSER
Refinement:
Amber
Structure from chemical shifts:
Fragment-based:
WeNMR CS-Rosetta
BMRB CS-Rosetta
Homology-based:
CS23D
Simshift
Torsion angles from chemical shifts:
Preditor
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Secondary structure from chemical shifts:
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MICS caps, β-turns
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PECAN
Flexibility from chemical shifts:
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Interactions from chemical shifts:
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Chemical shifts re-referencing:
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RDCs:
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Flexibility from structure:
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Methyl S2
B-factor
Molecular dynamics:
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From structure:
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CH3shift- Methyl
ArShift- Aromatic
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Proshift
PPM
CheShift-2- Cα
From sequence:
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Camcoil
Poulsen_rc_CS
Disordered proteins:
MAXOCC
Format conversion & validation:
CCPN
From NMR-STAR 3.1
Validate NMR-STAR 3.1
NMR sample preparation:
Protein disorder:
DisMeta
Protein solubility:
camLILA
ccSOL
Camfold
camGroEL
Zyggregator
Isotope labeling:
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Solid-state NMR:
sedNMR


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Old 11-24-2010, 09:51 PM
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Default High-throughput construction method for expression vector of peptides for NMR study s

High-throughput construction method for expression vector of peptides for NMR study suited for isotopic labeling.

Related Articles High-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.

PMID: 15166312 [PubMed - indexed for MEDLINE]



Source: PubMed
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