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

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

Strains and plasmids used in this study.

<p>Cm^R, Amp^R, Em^R, Tet^R and Kan^R: chloramphenicol, ampicillin, erythromycin, tetracyclin and kanamycin resistance.</p

Isomerization activity of rPpiA.

<p>A protease-free assay was used to measure PPIase activity. The prolyl <i>cis</i>→<i>trans</i> isomerisation of a tetrapeptide (Suc-Ala-Ala-Pro-Phe-2,4-difluoroanilide) was followed at 6°C by the decrease in absorbance at 246 nm (A_{246 nm}) as a function of time. Effects of PPIase addition (at a final concentration of 10 nM) or not (-, light grey line) were compared, using two different PPIases: rPpiA (grey line) or, as a positive control, hCyp18 (black line). The average of three independent experiments is shown.</p

Chaperone activity of rPpiA.

<p><b>A.</b> Citrate synthase (CS) was treated by concentrated guanidinium hydrochloride, and reactivation of unfolded CS was initiated by a 100-fold dilution into a buffer in the absence (□) or presence of rPpiA added at a CS∶rPpiA ratio of 1∶2 (▾). CS enzymatic activity was measured at the indicated time points, and recovered activity is shown (activity of native CS alone at the same concentration was set to 100%). <b>B.</b> CS refolding was followed like in A, in the absence (□) or presence of rPpiA at the following CS∶rPpiA ratios: 2∶1 (▪), 1∶1 (▴), 1∶2 (▾), 1∶5 (⧫), 1∶10 (×) and 1∶20 (+).</p

Protein sequence alignment between lactococcal PpiA protein and related cyclophilins.

<p>The sequences of some cyclophilins: <i>Homo sapiens</i> hCyp18 (Accession n° P62937), <i>E. coli</i> PpiA (P0AFL3), <i>S. pneumoniae</i> SlrA (NP_358273), and <i>L. lactis</i> PpiA (from strain IL1403: NP_266521.1 or strain MG1363: YP_001031737.1), are shown. Sequence alignment was performed using MultiAlin and manually improved to align the N-terminal hydrophobic domains of the three bacterial exported PPIases, and to take into account a previously published alignment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033516#pone.0033516-Hermans1" target="_blank">[12]</a>. Identical amino acids are marked with asterisks, and gaps with dash characters. The amino acids of the catalytic center of hCyp18 are marked in bold (in hCyp18 and all the proteins where they are conserved). The insertion sequence that is specific for <i>S. pneumoniae</i> SlrA compared to <i>E. coli</i> PpiA and hCyp18 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033516#pone.0033516-Hermans1" target="_blank">[12]</a>, and conserved in <i>L. lactis</i> PpiA, is boxed. The N-terminal hydrophobic sequence of lactococcal PpiA proteins is double underlined. PpiA peptides released by shaving treatment of MG1363 cells are underlined.</p

PpiA peptides released by shaving treatment of lactococcal cells.

<p>Six peptides identified by LCMS/MS were found to match with the same protein: its accession number, the gene name and protein function, <i>E</i>-values (for the whole protein and for each peptide) and coverage are indicated. In the first peptide, the amino acids in bold are conserved between <i>L. lactis</i> PpiA and hCyp18, and in the latter, they belong to the active center (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033516#pone-0033516-g001" target="_blank">Figure 1</a>).</p