title: Isotopic labeling of recombinant proteins expressed in the protozoan host Leishmania tarentolae.
abstract: 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 (15)N-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.
authors.affiliation: Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, Dortmund, Germany.
authors.forename: Anca
authors.initials: A
authors.lastname: Niculae
authors.forename: Peter
authors.initials: P
authors.lastname: Bayer
authors.forename: Ion
authors.initials: I
authors.lastname: Cirstea
authors.forename: Tim
authors.initials: T
authors.lastname: Bergbrede
authors.forename: Regina
authors.initials: R
authors.lastname: Pietrucha
authors.forename: Mathias
authors.initials: M
authors.lastname: Gruen
authors.forename: Reinhard
authors.initials: R
authors.lastname: Breitling
authors.forename: Kirill
authors.initials: K
authors.lastname: Alexandrov
mesh_terms: D000818:Animals; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007553:Isotope Labeling; D007891:Leishmania / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism*
chemical_list: D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins