A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli

A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli
Kit I. Tong, Masayuki Yamamoto and Toshiyuki Tanaka
Journal of Biomolecular NMR; 2008; 42(1); pp 59-67

Abstract:
A simple and user-friendly method of labeling protein selectively with amino acids in vivo is introduced. This technique does not require the use of transaminase-deficient or auxotrophic strains. By manipulating the product feedback inhibitory loops of the E. coli amino acid metabolic pathways and, if necessary, by using enzyme inhibitors, proteins were labeled efficiently in vivo even with amino acid types that are central to the metabolic pathways, such as glutamine. The sequential backbone resonance assignment of the Neh2 domain of Nrf2 transcriptional factor, an intrinsically disordered protein with high spectral degeneracy, was achieved using this labeling method.

Did you find this post helpful?

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Amino acid selective unlabeling for sequence specific resonance assignments in proteins Amino acid selective unlabeling for sequence specific resonance assignments in proteins Abstract Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative...	nmrlearner	Journal club	1	03-20-2012 12:42 AM
An efficient protocol for incorporation of an unnatural amino acid in perdeuterated recombinant proteins using glucose-based media An efficient protocol for incorporation of an unnatural amino acid in perdeuterated recombinant proteins using glucose-based media Abstract The in vivo incorporation of unnatural amino acids into proteins is a well-established technique requiring an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is incorporated at a position encoded by a TAG amber codon. Although this technology provides unique opportunities to engineer protein structures, poor protein yields are usually obtained in deuterated media, hampering its application in the protein NMR...	nmrlearner	Journal club	0	02-21-2012 03:40 AM
A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility Abstract An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract,...	nmrlearner	Journal club	0	10-05-2011 08:57 PM
Optimization of amino acid type-specific (13)C and (15)N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm. Optimization of amino acid type-specific (13)C and (15)N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm. Optimization of amino acid type-specific (13)C and (15)N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm. J Biomol NMR. 2010 Dec 18; Authors: Hefke F, Bagaria A, Reckel S, Ullrich SJ, Dötsch V, Glaubitz C, Güntert P We present a computational method for finding optimal labeling patterns for the backbone...	nmrlearner	Journal club	0	12-21-2010 01:00 PM
Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm Abstract We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273â??6279 (1982)), types of amino acids are labeled with 13C or/and 15N such that cross peaks between 13CO(i â?? 1) and 15NH(i) result only for pairs...	nmrlearner	Journal club	0	12-21-2010 02:14 AM
[NMR paper] Amino acid type selective isotope labelling of the multidrug ABC transporter LmrA for Amino acid type selective isotope labelling of the multidrug ABC transporter LmrA for solid-state NMR studies. Related Articles Amino acid type selective isotope labelling of the multidrug ABC transporter LmrA for solid-state NMR studies. FEBS Lett. 2004 Jun 18;568(1-3):117-21 Authors: Mason AJ, Siarheyeva A, Haase W, Lorch M, van Veen H, Glaubitz C The ABC transporter LmrA in Lactococcus lactis confers resistance to a wide range of antibiotics and cytotoxic drugs and is a functional homologue of P-glycoprotein. Recently, solid-state NMR...	nmrlearner	Journal club	0	11-24-2010 09:51 PM
An economical method for producing stable-isotope labeled proteins by the E. coli cel An economical method for producing stable-isotope labeled proteins by the E. coli cell-free system Abstract Improvement of the cell-free protein synthesis system (CF) over the past decade have made it one of the most powerful protein production methods. The CF approach is especially useful for stable-isotope (SI) labeling of proteins for NMR analysis. However, it is less popular than expected, partly because the SI-labeled amino acids used for SI labeling by the CF are too expensive. In the present study, we developed a simple and inexpensive method for producing an SI-labeled protein...	nmrlearner	Proteins	0	11-07-2010 02:47 PM
[NMR paper] Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis. Related Articles Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis. J Biomol NMR. 1995 Sep;6(2):129-34 Authors: Kigawa T, Muto Y, Yokoyama S For the application of multidimensional NMR spectroscopy to larger proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism drastically reduces the efficiency and selectivity...	nmrlearner	Journal club	0	08-22-2010 03:50 AM

« Previous Thread | Next Thread »

Posting Rules
You may not post new threads You may not post replies You may not post attachments You may not edit your posts BB code is On Smilies are On [IMG] code is On HTML code is On Trackbacks are Off Pingbacks are Off Refbacks are Off