A cost-effective approach for amino-acid-type selective isotope labeling of proteins expressed in Leishmania tarentolae

Silvie Trantírková¹, Jana Matulová¹, Volker Dötsch², Frank Löhr², Ion Cristea³, Kirill Alexandov³, Reinhard Breitling⁴, Julius Lukeš^1,5 and Lukáš Trantírek1^,5

¹Faculty of Biological Sciences, University of South Bohemia, České Budějovice, Czech Republic;
²Johann Wolfgang Goethe University, Franfurt am Main, Germany

³Institute for Physiology, Max-Plank Institute, Dortmund, Germany

⁴Jena Bioscience GmbH, Jena, Germany

⁵Biology Centre - Institute of Parasitology, Czech Academy of Sciences, České Budějovice, Czech Republic

e-mail: matulj00@bf.jcu.cz

We report a cost effecient approach for preparation of amino acid type selective (AATS), isotopically labeled proteins for nuclear magnetic resonance (NMR) spectroscopy using the Leishmania tarentolae expression system. The method is based on cultivation of the L. tarentolae inducible expression strain in a rich complex (BHI) 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 AATS isotope enrichment upon protein over-expression. Although low level isotope enrichment implies the decreased sensitivity of NMR experiment, the use of a rich complex medium leads to a two to three fold increase of the yield of the recombinant protein and, importantly, more than 10-fold reduction of the overall costs as compared to the recently established protocol for isotopic labeling using synthetic media (Niculae et al., 2006). We show that low-level enrichment does not compromise an NMR experiment and makes preparation of the recombinant proteins over-expressed in L. tarentolae economically viable. The method is demonstrated for selective labeling of ~27 kDa enhanced green fluorescent protein (EGFP) with ¹⁵N-labeled valine.

A. Niculae, P. Bayer, I. Cirstea, T. Bergbrede, R. Pietrucha, M. Gruen, R. Breitling & K. Alexandrov, Protein Expr. Purif., 48 (2006) 167-172.