A Cost-effective Amino-acid-type Selective Isotope Labeling of Proteins Expressed inLeishmania tarentolae

Silvie Foldynová-Trantírková, Jana Matulová, Volker Dötsch, Frank Löhr, Ion Cirstea, Kirill Alexandov, Reinhard Breitling, Julius Lukeš, Lukáš Trantírek
2009 Journal of Biomolecular Structure and Dynamics  
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
more » ... 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 overexpressed in L. tarentolae economically viable. The method is demonstrated by selective labeling of the ~27 kDa enhanced green fluorescent protein (EGFP) with 15 N-labeled valine.
doi:10.1080/07391102.2009.10507287 pmid:19385703 fatcat:u52orul6z5eetiu4jn6vjsyhiy