Isotopic labeling of recombinant proteins expressed in the protozoan host Leishmania tarentolae

• Published in: Protein expression and purification Vol. 48; no. 2; pp. 167 - 172
• Main Authors: Niculae, Anca, Bayer, Peter, Cirstea, Ion, Bergbrede, Tim, Pietrucha, Regina, Gruen, Mathias, Breitling, Reinhard, Alexandrov, Kirill
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-08-2006
• Subjects: 15N-labeling; Animals; Eukaryotic expression system; Gene Expression; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Isotope Labeling; Leishmania - genetics; Leishmania - metabolism; Leishmania tarentolae; Magnetic Resonance Spectroscopy; Nitrogen Isotopes; Recombinant protein; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; 15N-labeling; Recombinant protein; Eukaryotic expression system
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2006.04.006

Isotope labeling of recombinant proteins is a prerequisite for application of nuclear magnetic resonance spectroscopy (NMR) for the characterization of the three-dimensional structures and dynamics of proteins. Overexpression of isotopically labeled proteins in bacterial or yeast host organisms has several drawbacks. In this work, we tested whether the recently described eukaryotic protein expression system based on the protozoa
Leishmania tarentolae could be used for production of amino acid specific
15N-labeled recombinant proteins. Using synthetic growth medium we were able to express in
L. tarentolae and purify to homogeneity (15)N-valine labeled Enchanced Green Fluorescent Protein (EGFP) with the final yield of 5.7
mg/liter of suspension culture. NMR study of isolated EGFP illustrated the success of the labeling procedure allowing identification of all 18 valine residues of the protein in the HSQC spectrum. Our results demonstrate the suitability of the
L. tarentolae expression system for production of isotopically labeled proteins.