Purification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolae

The rapid and inexpensive production of high-quality eukaryotic proteins in recombinant form still remains a challenge in structural biology. Here, a protein-expression system based on the protozoan Leishmania tarentolae was used to produce human Cu/Zn superoxide dismutase (SOD1) in recombinant form. Sequential integration of the SOD1 expression cassettes was demonstrated to lead to a linear increase in expression levels to up to 30 mg per litre. Chromatographic purification resulted in 90% pure recombinant protein, with a final yield of 6.5 mg per litre of culture. The protein was crystallized and the structures of two new crystal forms were determined. These results demonstrate the suitability of the L. tarentolae expression system for structural research

Entwicklung von Methoden zur Manipulation der Genexpression im Protozoon Leishmania tarentolae\textit {Leishmania tarentolae}

Um ein hohes Niveau der Überexpression heterologer Proteine in $\textit {L. tarentolae}$ zu erreichen, bestand der Ansatz in der Steigerung der Menge heterologer mRNA. Dies sollte entweder durch eine Reduktion der Abbau- oder eine Steigerung der Produktionsrate der mRNA geschehen. Die Reduktion der Abbaurate basiert auf dem derzeit aktuellen Modell der Genexpression der $\it {Trypanosomatidae}$, für welche angenommen wird, dass die 3´UTR des proteinkodierenden Gens die Stabilität der mRNA bestimmen. Dazu wurden zufällig ausgewählte Sequenzen aus dem $\textit {L. tarentolae}$-Genom auf ihre Eignung als 3´UTR eines proteinkodierenden Gens zu funktionieren überprüft. In einem zweiten Ansatz konnte eine Erhöhung in der mRNA-Produktion durch eine Steigerung der Kopienzahl transkribierter Gene erreicht werden. Zu diesem Zweck wurde der induzierbare $\textit {L. tarentolae}$ Stamm genutzt. Dazu wurde ein artifizielles Episom für induzierbare Proteinexpression konstruiert

Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1

Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected Spodoptera frugiperda insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from Escherichia coli, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the in vitro liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes

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

Deregulation of Hepatic Mek1/2–Erk1/2 Signaling Module in Iron Overload Conditions

The liver, through the production of iron hormone hepcidin, controls body iron levels. High liver iron levels and deregulated hepcidin expression are commonly observed in many liver diseases including highly prevalent genetic iron overload disorders. In spite of a number of breakthrough investigations into the signals that control hepcidin expression, little progress has been made towards investigations into intracellular signaling in the liver under excess of iron. This study examined hepatic signaling pathways underlying acquired and genetic iron overload conditions. Our data demonstrate that hepatic iron overload associates with a decline in the activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) kinase (Mek1/2) pathway by selectively affecting the phosphorylation of Erk1/2. We propose that Mek1/2-Erk1/2 signaling is uncoupled from iron-Bmp-Smad-mediated hepcidin induction and that it may contribute to a number of liver pathologies in addition to toxic effects of iron. We believe that our findings will advance the understanding of cellular signaling events in the liver during iron overload of different etiologies

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

Mechanistic Insights into Specificity, Activity, and Regulatory Elements of the Regulator of G-protein Signaling (RGS)-containing Rho-specific Guanine Nucleotide Exchange Factors (GEFs) p115, PDZ-RhoGEF (PRG), and Leukemia-associated RhoGEF (LARG)*

The multimodular guanine nucleotide exchange factors (GEFs) of the Dbl family mostly share a tandem Dbl homology (DH) and pleckstrin homology (PH) domain organization. The function of these and other domains in the DH-mediated regulation of the GDP/GTP exchange reaction of the Rho proteins is the subject of intensive investigations. This comparative study presents detailed kinetic data on specificity, activity, and regulation of the catalytic DH domains of four GEFs, namely p115, p190, PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG). We demonstrate that (i) these GEFs are specific guanine nucleotide exchange factors for the Rho isoforms (RhoA, RhoB, and RhoC) and inactive toward other members of the Rho family, including Rac1, Cdc42, and TC10. (ii) The DH domain of LARG exhibits the highest catalytic activity reported for a Dbl protein till now with a maximal acceleration of the nucleotide exchange by 107-fold, which is at least as efficient as reported for GEFs specific for Ran or the bacterial toxin SopE. (iii) A novel regulatory region at the N terminus of the DH domain is involved in its association with GDP-bound RhoA monitored by a fluorescently labeled RhoA. (iv) The tandem PH domains of p115 and PRG efficiently contribute to the DH-mediated nucleotide exchange reaction. (v) In contrast to the isolated DH or DH-PH domains, a p115 fragment encompassing both the regulator of G-protein signaling and the DH domains revealed a significantly reduced GEF activity, supporting the proposed models of an intramolecular autoinhibitory mechanism for p115-like RhoGEFs

A novel in vitro assay to study chondrocyte-to-osteoblast transdifferentiation

Purpose!#!Endochondral ossification, which involves transdifferentiation of chondrocytes into osteoblasts, is an important process involved in the development and postnatal growth of most vertebrate bones as well as in bone fracture healing. To study the basic molecular mechanisms of this process, a robust and easy-to-use in vitro model is desirable. Therefore, we aimed to develop a standardized in vitro assay for the transdifferentiation of chondrogenic cells towards the osteogenic lineage.!##!Methods!#!Murine chondrogenic ATDC5 cells were differentiated into the chondrogenic lineage for seven days and subsequently differentiated towards the osteogenic direction. Gene expression analysis of pluripotency, as well as chondrogenic and osteogenic markers, cell-matrix staining, and immunofluorescent staining, were performed to assess the differentiation. In addition, the effects of Wnt3a and lipopolysaccharides (LPS) on the transdifferentiation were tested by their addition to the osteogenic differentiation medium.!##!Results!#!Following osteogenic differentiation, chondrogenically pe-differentiated cells displayed the expression of pluripotency and osteogenic marker genes as well as alkaline phosphatase activity and a mineralized matrix. Co-expression of Col2a1 and Col1a1 after one day of osteogenic differentiation indicated that osteogenic cells had differentiated from chondrogenic cells. Wnt3a increased and LPS decreased transdifferentiation towards the osteogenic lineage.!##!Conclusion!#!We successfully established a rapid, standardized in vitro assay for the transdifferentiation of chondrogenic cells into osteogenic cells, which is suitable for testing the effects of different compounds on this cellular process

Nucleotide-independent extraction of Rac1^Ic from the liposomes by GDI1.

<p>(<b>A</b>) GST-GDI1 pull-down of Rac1^Ic but not of Rac1<i>^Ec</i>. Input is the total mixture of beads and proteins, and output is the pull-down (PD). (<b>B</b>) Liposome binding of Rac1^Ic but not of Rac1<i>^Ec</i>. In the liposome sedimentation assay, Rac1^Ic efficiently binds to liposomes in the absence of GDI1 and independent of whether it was loaded with GDP or GppNHp, a non-hydrolysable GTP analog. Rac1<i>^Ec</i> failed to bind to liposomes under the same conditions. (<b>C</b>) Preferential binding Rac1^Ic to GDI1 than to liposomes. GDI1 binds to both GDP-bound and GppNHp-bound Rac1^Ic proteins and prevents their association with the liposomes. (<b>D</b>, <b>E</b>) GDI1 efficiently extracted GDP-bound Rac1^Ic from the liposomes and to a lower extend also Rac1^Ic-GppNHp. Same amount of GDP-bound and GppNHp-bound forms of Rac1^Ic associated with the liposomes were prepared before incubation with 5-fold molar excess of GDI1 and sedimentation at 20,000x<i>g</i> (<b>D</b>). Using increasing molar excess of GDI1 (2-, 5-, 10-, 15- and 20-fold) showed that higher concentrations of GDI1 are required to extract Rac1^Ic-GppNHp from the liposomes to supernatants in comparison to Rac1^Ic-GDP (<b>E</b>). CBB, coomassie brilliant blue; <i>Ec</i>, <i>E. coli</i>; Ic, insect cells; P, liposome pellet; S, supernatant.</p