Phosphoinositide 3-kinase γ mediates Jun kinase activation via its lipid-kinase activity

Phosphoinositide 3-kinases (PI3K) are bifunctional enzymes expressing lipid- and protein-kinase activities. PI3K lipid kinase is involved in the control of cell motility, survival and other cellular reactions. Recent data point to specific signaling functions of PI3K protein-kinase activities. The PI3K species g has been shown to stimulate major signaling paths to the protein kinases Erk, JNK and PKB, in COS-7 cells. The present work was aimed to decipher the signaling reactions involved in the control of JNK activity by PI3Kg. Using functionally defined mutants we demonstrate an essential role of PI3Kg lipid-kinase activity for JNK stimulation. Together with recent findings these data evidence differential effects of PI3Kg protein- and lipidkinase activities on Erk, JNK and PKB signaling pathways

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 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

Non-pathogenic trypanosomatid protozoa as a platform for protein research and production

All currently existing eukaryotic protein expression systems are based on autonomous life forms. To exploit the potential practical benefits associated with parasitic organisms we have developed a new protein expression system based on Leishmania tarentolae (Trypanosomatidae), a protozoan parasite of lizards. To achieve strong transcription, the genes of interest were integrated into the small subunit ribosomal RNA gene. Expression levels obtained were up to 30mg of recombinant protein per liter of suspension culture and increased linearly with the number of integrated gene copies. To assess the system's potential for production of post-translationally modified proteins, we have expressed human erythropoietin in L. tarentolae. The recombinant protein isolated from the culture supernatants was biologically active, natively processed at the N-terminus, and N-glycosylated. The N-glycosylation was exceptionally homogenous, with a mammalian-type biantennary oligosaccharide and the Man(3)GlcNAc(2) core structure accounting for >90% of the glycans present. L. tarentolae is thus the first described biotechnologically useful unicellular eukaryotic organism producing biantennary fully galactosylated, core-alpha-1,6-fucosylated N-glycans. (C) 2002 Elsevier Science (USA). All rights reserved

Non-pathogenic trypanosomatid protozoa as a platform for protein research and production

All currently existing eukaryotic protein expression systems are based on autonomous life forms. To exploit the potential practical benefits associated with parasitic organisms we have developed a new protein expression system based on Leishmania tarentolae (Trypanosomatidae), a protozoan parasite of lizards. To achieve strong transcription, the genes of interest were integrated into the small subunit ribosomal RNA gene. Expression levels obtained were up to 30mg of recombinant protein per liter of suspension culture and increased linearly with the number of integrated gene copies. To assess the system's potential for production of post-translationally modified proteins, we have expressed human erythropoietin in L. tarentolae. The recombinant protein isolated from the culture supernatants was biologically active, natively processed at the N-terminus, and N-glycosylated. The N-glycosylation was exceptionally homogenous, with a mammalian-type biantennary oligosaccharide and the Man(3)GlcNAc(2) core structure accounting for >90% of the glycans present. L. tarentolae is thus the first described biotechnologically useful unicellular eukaryotic organism producing biantennary fully galactosylated, core-alpha-1,6-fucosylated N-glycans. (C) 2002 Elsevier Science (USA). All rights reserved