NDLI: Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing

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Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing

Measurement of 15N relaxation rates in perdeuterated proteins by TROSY-based methods

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Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing

Content Provider	PubMed Central
Author	Minato, Yuichi Ueda, Takumi Asako, Machiyama Shimada, Ichio Iwaï, Hideo
Abstract	Segmental isotopic labeling is a powerful labeling tool to facilitate NMR studies of larger proteins by not only alleviating the signal overlap problem but also retaining features of uniform isotopic labeling. Although two approaches, expressed protein ligation (EPL) and protein trans-splicing (PTS), have been mainly used for segmental isotopic labeling, there has been no single example in which both approaches have been directly used with an identical protein. Here we applied both EPL and PTS methods to a 140 kDa dimeric multi-domain protein E. coli CheA, and successfully produced the ligated CheA dimer by both approaches. In EPL approach, extensive optimization of the ligation sites and the conditions were required to obtain sufficient amount for an NMR sample of CheA, because CheA contains a dimer forming domain and it was not possible to achieve high reactant concentrations (1–5 mM) of CheA fragments for the ideal EPL condition, thereby resulting in the low yield of segmentally labelled CheA dimer. PTS approach sufficiently produced segmentally labeled ligated CheA in vivo as well as in vitro without extensive optimizations. This is presumably because CheA has self-contained domains connected with long linkers, accommodating a seven-residue mutation without loss of the function, which was introduced by PTS to achieve the high yield. PTS approach was less laborious than EPL approach for the routine preparation of segmentally-isotope labeled CheA dimer. Both approaches remain to be further developed for facilitating preparations of segmental isotope-labelled samples without extensive optimizations for ligation.
Related Links	http://dx.doi.org/10.1007/s10858-012-9628-3
Ending Page	207
Page Count	17
Starting Page	191
File Format	PDF
ISSN	09252738
e-ISSN	15735001
Journal	Journal of Biomolecular Nmr
Issue Number	3
Volume Number	53
Language	English
Publisher	Springer Netherlands
Publisher Date	2012-07-01
Access Restriction	Open
Rights Holder	Springer Netherlands
Subject Keyword	Spectroscopy Biochemistry Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Spectroscopy Biochemistry

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Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing

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