A cost-effective amino-acid-type selective isotope labeling of proteins expressed in Leishmania tarentolae.
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 approximately 27 kDa enhanced green fluorescent protein (EGFP) with 15N-labeled valine.
Foldynová-Trantirková
Silvie
S
University of South Bohemia, Ceské Budejovice, Czech Republic.
Matulová
Jana
J
Dötsch
Volker
V
Löhr
Frank
F
Cirstea
Ion
I
Alexandov
Kirill
K
Breitling
Reinhard
R
Lukes
Julius
J
Trantírek
Lukás
L
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D003362:Cost-Benefit Analysis; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007553:Isotope Labeling / Q000191:economics / Q000379:methods*; D007891:Leishmania / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015203:Reproducibility of Results; D014162:Transfection
D000596:Amino Acids; D009587:Nitrogen Isotopes; D011506:Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins