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

Abstract

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 speciWc 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 Wnal yield of 5.7 mg/liter of suspension culture. NMR study of isolated EGFP illustrated the success of the labeling procedure allowing identiWcation 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. © 2006 Elsevier Inc. All rights reserved. Keywords: 15 N-labeling; Recombinant protein; Eukaryotic expression system Nuclear magnetic resonance spectroscopy (NMR) 1 is one of two existing methods that allow determination of protein structure at atomic resolution. A majority of NMR techniques in biology require isotopic labeling ( 2 H, 13 C, and/or 15 N) of recombinant proteins. Currently, most isotopically labeled recombinant proteins are expressed heterologously in Escherichia coli. Despite its obvious advantages such as rapid growth, developed methods of protein expression and cheapness of cultivation E. coli has a range of shortcomings that limits its utility in protein studies. The most prominent problem relates to ineYciency of E. coli to assist folding of eukaryotic polypeptides producing only ca. 15% of eukaryotic proteins in their active form We recently described a new protein expression system based on the non-pathogenic trypanosomatid Leishmani