On the use of electrochemical techniques to monitor free oxide content in molten fluoride media

The electrochemical behaviour of oxide ions has been studied in fluoride melts(LiF/NaF eutectic) by cyclic voltammetry, square wave voltammetry and chronopotentiometry. The purpose is to determine whether these techniques can be used for titration of free oxide ions (O2-) in molten fluorides released by lithium oxide additions. Cyclic voltammetry is shown to be unsuitable for this purpose due to oxygen bubbling disturbing the oxidation peak, while square wave voltammetry is far more appropriate because the observed signal is a well defined oxidation peak with a height proportional to the oxide content. Thus, the present work is focused on a strategy of oxide ions titration by square wave voltammetry. In addition, this work allows assessing that the electrochemical reduction of oxide ions proceeds by diffusion of these species, and the O2- diffusion coefficient is estimated by chronopotentiometry

PpiA, a Surface PPIase of the Cyclophilin Family in Lactococcus lactis

Background: Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes. Results: In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H2O2) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H2O2. Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. Conclusions: Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displaye

Cloning and in silico characterization of two signal peptides from Pediococcus pentosaceus and their function for the secretion of heterologous protein in Lactococcus lactis.

Fifty signal peptides of Pediococcus pentosaceus were characterized by in silico analysis and, based on the physicochemical analysis, (two potential signal peptides Spk1 and Spk3 were identified). The coding sequences of SP were amplified and fused to the gene coding for green fluorescent protein (GFP) and cloned into Lactococcus lactis pNZ8048 and pMG36e vectors, respectively. Western blot analysis indicated that the GFP proteins were secreted using both heterologous SPs. ELISA showed that the secretion efficiency of GFP using Spk1 (0.64 μg/ml) was similar to using Usp45 (0.62 μg/ml) and Spk3 (0.58 μg/ml)

Amélioration de la secrétion hétérologue chez lactococcus lactis

La bactérie lactique Lactococcus lactis est un hôte intéressant pour produire, sécréter et purifier des protéines hétérologues. L INRA a construit un système d expression et une souche hôte mutée dans l'unique protéase extracellulaire HtrA et où les protéines exportées sont stables, d où un meilleur rendement et un avantage concurrentiel. Le système de production INRA est développé en partenariat avec GTP Technology dans le cadre de ma thèse CIFRE. Pour améliorer la souche hôte, j ai recherché des leviers susceptibles de compenser les fonctions de HtrA, résistance au stress thermique et contrôle qualité des protéines exportées. J ai sélectionné un suppresseur en multicopie de la thermo-sensibilité du mutant htrA : de fonction inconnue, son effet sur la production de protéines doit être testé. J ai aussi caractérisé un facteur de repliement de surface, une PPIase, qui n a pas d effet sur les protéines testées, et qui aurait un rôle de ménage constitutif. Enfin, j ai étudié une protéine induite dans le mutant htrA en conditions normales: c est une protéine de stress de paroi, induite dans la souche sauvage par des antibiotiques qui ciblent la biosynthèse du peptidoglycane, et essentielle à la survie à la bacitracine. Ainsi, l absence de HtrA perturbe non seulement les protéines d enveloppe mais aussi la paroi. L étude de cette nouvelle fonction de HtrA et du rôle de la protéine induite dans la survie va être poursuivie. Parallèlement, j ai aussi développé l utilisation du système INRA chez GTP-Technology. J ai mis au point la production d une nucléase utile en biologie moléculaire pour clarifier des extraits bactériens, avec un rendement de 200mg/L en mini-fermenteurs.The lactic acid bacterium Lactococcus lactis is an interesting host to produce, secrete and purify heterologous proteins. INRA built up an expression system and a host strain inactivated for the unique extracellular HtrA protease and in which exported proteins are stable, leading to a better yield and a competitive advantage. INRA system development in partnership with GTP Technology is the objective of my PhD thesis (CIFRE fellowship). To improve the host strain, positive factors able to compensate for HtrA functions: heat shock resistance and the quality control of exported proteins, were looked for. A multicopy suppressor of htrA mutant thermosensitivity was selected: its function is unknown and its effect on heterologous protein production has to be tested. An exported folding factor, a PPIase, was also caracterized, but it had no effect on the proteins that have been tested, suggesting a constitutive housekeeping function. At last, a protein induced in htrA mutant under normal conditions was studied. It is a cell wall stress protein, both induced in the wild type strain by several antibiotics targeting peptidoglycan biosynthesis and essential for cell viability when cells are exposed to bacitracin. Thus, the absence of HtrA not only disturbs envelope proteins, but also the cell wall. This new HtrA function and of the role of the induced protein on cell survival will be further studied. In parallel, the use of the INRA expression system at GTP Technology was also developed. The conditions for the production of a nuclease useful in molecular biology to clarify cell extracts were optimised, and a protein yield of about 200mg/L in mini-fermentors could be reached.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF