3 research outputs found
Artificial linear episome-based protein expression system for protozoon Leishmania tarentolae
The trypanosomatid protozoon Leishmania tarentolae is a well-established model organism for studying causative agents of several tropical diseases that was more recently developed as a host for recombinant protein production. Although several expression architectures based on foreign RNA polymerases have been established for this organism, all of them rely on integration of the expression cassette into the genome. Here, we exploit a new type of expression architecture based on linear elements. These expression vectors were propagated in Escherichia coli as circular plasmids and converted into linear episomes with telomere-like structures prior to transfection of L. tarentolae. Overexpression of recombinant proteins in transgenic organisms exceeding 10% of total cellular protein, one of the highest overexpression levels obtained in a eukaryotic organism for a cytosolic protein. We show that the linear elements are stably propagated in L. tarentolae cells over long periods of time (>90 generations) without major changes in structure or expression yields. Overexpressing cultures can be obtained without clonal selection of the transfected cells. To establish the utility of the developed system for protein production in a parallelized format, we expressed 37 cytosolic, peripheral, and membrane proteins as fusions with EGFP in L. tarentolae using linear vectors. We detected the expression of 30 of these targets and describe the preparative purification of two arbitrarily selected proteins
Fluorescence spectroscopy of roGFP2-based redox probes responding to various physiologically relevant oxidant species in vitro
This article contains representative fluorescence excitation spectra of roGFP2-based probes used for ratiometric analysis of redox changes as presented in the article "Systematic in vitro assessment of responses of roGFP2-based probes to physiologically relevant oxidant species" [1]. The recombinant probes roGFP2, roGFP2-Orp1, and Grx1-roGFP2 were exposed to various oxidative and nitrosative species, including hydrogen peroxide (H2O2), aldrithiol-2 (AT-2), glutathione disulfide (GSSG), hypochlorous acid (HOCl), S-nitrosoglutathione (GSNO), peroxynitrite (ONOO−), potassium polysulfide (K2Sx), spermine NONOate (SperNO), and diethyl amino NONOate (DeaNO) at different molar ratios. Fluorescence excitation spectra of the probes were recorded in the excitation wavelength range between 350 and 500 nm and for a total of 60 min. Analysis and interpretation of the data is presented in an associated article [1]
Structures of RabGGTase–substrate/product complexes provide insights into the evolution of protein prenylation
Post-translational isoprenylation of proteins is carried out by three related enzymes: farnesyltransferase, geranylgeranyl transferase-I, and Rab geranylgeranyl transferase (RabGGTase). Despite the fact that the last one is responsible for the largest number of individual protein prenylation events in the cell, no structural information is available on its interaction with substrates and products. Here, we present structural and biophysical analyses of RabGGTase in complex with phosphoisoprenoids as well as with the prenylated peptides that mimic the C terminus of Rab7 GTPase. The data demonstrate that, unlike other protein prenyl transferases, both RabGGTase and its substrate RabGTPases completely ‘outsource' their specificity for each other to an accessory subunit, the Rab escort protein (REP). REP mediates the placement of the C terminus of RabGTPase into the active site of RabGGTase through a series protein–protein interactions of decreasing strength and selectivity. This arrangement enables RabGGTase to prenylate any cysteine-containing sequence. On the basis of our structural and thermodynamic data, we propose that RabGGTase has evolved from a GGTase-I-like molecule that ‘learned' to interact with a recycling factor (GDI) that, in turn, eventually gave rise to REP