A cost-effective amino-acid-type selective isotope labeling of proteins expressed in leishmania tarentolae

Foldynova-Trantirkova, S., Matulova, J., Dotsch, V., Lohr, F., Cirstea, I., Alexandov, K., Breitling, R., Lukes, J. and Trantirek, L. (2009) A cost-effective amino-acid-type selective isotope labeling of proteins expressed in leishmania tarentolae. Journal of Biomolecular Structure & Dynamics, 26 6: 755-762.


Author Foldynova-Trantirkova, S.
Matulova, J.
Dotsch, V.
Lohr, F.
Cirstea, I.
Alexandov, K.
Breitling, R.
Lukes, J.
Trantirek, L.
Title A cost-effective amino-acid-type selective isotope labeling of proteins expressed in leishmania tarentolae
Journal name Journal of Biomolecular Structure & Dynamics   Check publisher's open access policy
ISSN 0739-1102
Publication date 2009-06
Year available 2009
Sub-type Article (original research)
Volume 26
Issue 6
Start page 755
End page 762
Total pages 8
Editor Ramaswamy H. Sarma
Place of publication Schenectady, NY, U.S.A.
Publisher Adenine Press
Language eng
Subject 060107 Enzymes
970106 Expanding Knowledge in the Biological Sciences
CX
Abstract We report a cost efficient approach for amino-acid-type selective isotope labeling of proteins expressed in Leishmania tarentolae. The method provides an economically advantageous alternative to recently established protocol for isotopic labeling using expensive synthetic media. The method is based on cultivation of the L. tarentolae expression strain in a cheap complex medium supplemented with labeled amino acid(s). In this protocol, a labeled amino acid is deliberately diluted in the medium of undefined composition, which leads to a low-level isotope enrichment upon protein over-expression. The economic advantage of the protocol is achieved by avoiding large volumes of expensive synthetic medium. Decreased sensitivity of a NMR experiment due to low-level isotope enrichment is compensated by a five- to seven-fold increase of the yield of the recombinant protein in complex medium as compared to that in the synthetic medium. In addition, the decreased sensitivity can be compensated by using a higher magnetic field, cryo-detection system or higher number of transients during the NMR data acquisition. We show that enrichment as low as 5% does not compromise a NMR experiment and makes preparation of the recombinant proteins over-expressed in L. tarentolae economically viable. The method is demonstrated by selective labeling of the ~27 kDa enhanced green fluorescent protein (EGFP) with 15N-labeled valine.
Keyword NMR
Isotope labeling
Protein expression
Leishmania
Low-level enrichment
Infected Insect Cells
Pichia-Pastoris
Recombinant Proteins
Macromolecules
Transcription
Spectroscopy
Sensitivity
Relaxation
Enrichment
Q-Index Code CX
Q-Index Status Provisional Code

Document type: Journal Article
Sub-type: Article (original research)
Collection: Institute for Molecular Bioscience - Publications
 
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Created: Tue, 17 Nov 2009, 12:08:11 EST