Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor

<p>Abstract</p> <p>Background</p> <p>Staphylococcal (or micrococcal) nuclease or thermonuclease (SNase or Nuc) is a naturally-secreted nucleic acid degrading enzyme that participates in <it>Staphylococcus aureus </it>spread in the infected host. Purified Nuc protein can be used as an exogenous reagent to clear cellular extracts and improve protein purification. Here, a recombinant form of Nuc was produced and secreted in a Gram-positive host, <it>Lactococcus lactis</it>, and purified from the culture medium.</p> <p>Results</p> <p>The gene segment corresponding to the <it>S. aureus </it>nuclease without its signal peptide was cloned in an expression-secretion vector. It was then fused to a lactococcal sequence encoding a signal peptide, and expressed under the control of a lactococcal promoter that is inducible by zinc starvation. An <it>L. lactis </it>subsp <it>cremoris </it>model strain (MG1363) transformed with the resulting plasmid was grown in either of two media (GM17v and CDM) that are free of animal compounds, allowing GMP (Good Manufacturing Practice) production. Induction conditions (concentration of the metal chelator EDTA and timing of addition) in small-scale pH-regulated fermentors were optimized using LacMF (Lactis Multi-Fermentor), a home-made parallel fermentation control system able to monitor 12 reactors simultaneously. Large amounts of recombinant Nuc (rNuc) were produced and secreted in both media, and rNuc was purified from GM17v medium in a single-step procedure.</p> <p>Conclusions</p> <p>In <it>L. lactis</it>, rNuc production and secretion were optimal after induction by 0.5 mM EDTA in small scale (200 mL) GM17v exponential phase cultures (at an OD₆₀₀of 2), leading to a maximal protein yield of 210 mg per L of culture medium. Purified rNuc was highly active, displaying a specific activity of 2000 U/mg.</p

Conjugation of genetically-engineered protein phosphatases to magnetic particles for okadaic acid detection

This work presents the functional characterisation of a protein phosphatase 2A (PP2A) catalytic subunit obtained by genetic engineering and its conjugation to magnetic particles (MPs) via metal coordination chemistry for the subsequent development of assays for diarrheic lipophilic marine toxins. Colorimetric assays with free enzyme have allowed the determination of the best enzyme activity stabiliser, which is glycerol at 10%. They have also demonstrated that the recombinant enzyme can be as sensitive towards okadaic acid (OA) (LOD=2.3μg/L) and dinophysistoxin-1 (DTX-1) (LOD=15.2μg/L) as a commercial PP2A and, moreover, it has a higher operational stability, which makes possible to perform the protein phosphatase inhibition assay (PPIA) with a lower enzyme amount. Once conjugated to MPs, the PP2A catalytic subunit still retains its enzyme activity and it can also be inhibited by OA (LOD=30.1μg/L)

Tetrathiafulvalene-phosphine-based iron and ruthenium carbonyl complexes: Electrochemical and EPR studies

The radical cation of the redox active ligand 3,4-dimethyl-3,4-bis-(diphenylphosphino)-tetrathiafulvalene ( P2) has been chemically and electrochemically generated and studied by EPR spectroscopy. Consistent with DFT calculations, the observed hyperfine structure (septet due to the two methyl groups) indicates a strong delocalization of the unpaired electron on the central S2CCS2 part of the tetrathiafulvalene (TTF) moiety and zero spin densities on the phosphine groups. In contrast with the ruthenium(0) carbonyl complexes of P2 whose one-electron oxidation directly leads to decomplexation and produces P2+, one-electron oxidation of [Fe( P2)(CO)3] gives rise to the metal-centered oxidation species [Fe(I)( P2)(CO)3], characterized by a coupling with two 31P nuclei and a rather large g-anisotropy. The stability of this complex is however modest and, after some minutes, the species resulting from the scission of a P–Fe bond is detected. Moreover, in presence of free ligand, [Fe(I)( P2)(CO)3] reacts to give the complex [Fe(I)( P2)2(CO)] containing two TTF fragments. The two-electron oxidation of [Fe( P2)(CO)3] leads to decomplexation and to the P2+ spectrum. Besides EPR spectroscopy, cyclic voltammetry as well as FTIR spectroelectrochemistry are used in order to explain the behaviour of [Fe( P2)(CO)3] upon oxidation. This behaviour notably differs from that of the Ru(0) counterpart. This difference is tentatively rationalized on the basis of structural arguments