ABSTRACT
Nucleoside phosphates can bind to many functional proteins like G-proteins or other GTP-binding proteins in signal transduction or translation processes. Till now internalization of nucleoside phosphates into live cells remains a challenge. We study the internalization of a fluorescent-labelled deoxyuridine triphosphate into HeLa cells and other adhesion and suspension cells. We use different cell-penetrating peptides and a cocktail suitable for formation of non-covalent complexes with the nucleotide. Internalization is observed by fluorescence microscopy, and the uptake efficiency is quantitatively estimated by fluorescence spectroscopy. The applied concentrations of CPPs and the cocktail were checked on cell viability (MTT test) and membrane integrity (bioluminescence test with peptidyl-luciferin), indicating that the CPPs and the complexes with the nucleotide are cytotoxic above certain concentrations. These concentrations depend on CPP and cell type and are the limiting factors for the cargo uptake.
Subject(s)
Cell-Penetrating Peptides/metabolism , Nucleotides/metabolism , Amino Acid Sequence , Animals , Biological Transport , Cell Adhesion , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Survival/drug effects , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/toxicity , Humans , Mice , Microscopy, Fluorescence , Molecular Sequence Data , Protein BindingABSTRACT
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.
Subject(s)
Gene Expression , Leishmania/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Animals , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Isotope Labeling , Leishmania/genetics , Magnetic Resonance Spectroscopy , Nitrogen IsotopesABSTRACT
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 30 mg 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 homogeneous, 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.
