Related ArticlesA Streamlined Method for Preparing Split Intein for NMR Study.
Protein Expr Purif. 2014 Apr 18;
Authors: Lee YZ, Lee YT, Lin YJ, Chen YJ, Sue SC
Abstract
A protein ligase, intein, mediates a protein-splicing reaction. It can be split into two complementary fragments and reconstituted as a whole intein scaffold to perform protein trans-splicing. To understand the association of intein fragments and the splicing mechanism, it is necessary to produce a large quantity of split intein for structural study. Conventionally, two fragments are prepared separately and assembled in solution, but severe aggregation of intein fragments occurs, and precise control of the relative concentration of each fragment is difficult. Here, we present a streamlined method to incorporate a circular permutation concept into the production of split intein. By circular permutation of the native split Nostoc punctiforme DnaE intein (NpuInt), a new backbone opening is relocated to the native split site at residue 102. As the protein splicing activity is preserved, the expressed NpuInt can immediately self-cleave into a two-piece split NpuInt. Because of a tight association between the two complementary fragments, split NpuInt can be purified in one step. The idea is simple and applicable to other split inteins. Employing the new preparation, we use NMR spectra to assign the backbone and side chain resonances for the native split NpuInt.
PMID: 24751877 [PubMed - as supplied by publisher]
(1)H, (13)C, and (15)N NMR assignments of the Pyrococcus abyssi DNA polymerase II intein.
(1)H, (13)C, and (15)N NMR assignments of the Pyrococcus abyssi DNA polymerase II intein.
(1)H, (13)C, and (15)N NMR assignments of the Pyrococcus abyssi DNA polymerase II intein.
Biomol NMR Assign. 2011 Apr 26;
Authors: Liu J, Du Z, Albracht CD, Naidu RO, Mills KV, Wang C
Protein splicing is a precise post-translational process mediated by inteins. Inteins are intervening proteins that cleave themselves from a precursor protein while joining the flanking sequences. Here we report the (15)N, (13)C, and (1)H chemical shift assignments of the intein...
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[NMR paper] Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically l
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Nat Biotechnol. 2005 Jun;23(6):736-40
Authors: Züger S, Iwai H
Segmental isotopic labeling of proteins using protein ligation is a recently established in vitro method for incorporating isotopes into one domain or region of a protein to reduce the complexity of NMR spectra, thereby facilitating...
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[NMR paper] High-throughput construction method for expression vector of peptides for NMR study s
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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...
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The size limit for protein NMR spectroscopy in solution arises in large part from line broadening caused by slow molecular tumbling. One way to alleviate this problem is to increase the effective tumbling rate by reducing the viscosity of the solvent. Because proteins generally require an aqueous environment to...
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Brain Res Brain Res Protoc. 2000 Apr;5(2):182-9
Authors: Hirai H, Yoshioka K, Yamada K
Nonradioactive 31P-NMR spectroscopy has previously been used for the study of protein phosphorylations. However, the procedures does not seem to be easy for non-experts of this field, hence, this approach has not been widely used. We introduce here a simple protocol with...
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J Magn Reson. 1998 Sep;134(1):154-7
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A new method for NMR spectra acquisition of paramagnetic proteins is described, based on the simple use of homonuclear broadband decoupling of the diamagnetic region. Several advantages are associated with this method which was applied to one-dimensional spectra, to 1D...
A Streamlined Method for Preparing Split Intein for NMR Study.
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