Expression of soluble recombinant proteins in a cell-free system using a 96-well format

For structural and functional genomics programs, new high-throughput methods to obtain well-expressing and highly soluble proteins are essential. Here, we describe a rapid procedure to express recombinant proteins in an Escherichia coli cell-free system using a 96-well format. The identification of soluble proteins is performed by the Dot Blot procedure using an anti-His tag antibody. The applications and the automation of this method are described.     

Identification of biologic markers of the premature rupture of fetal membranes: proteomic approach

In obstetrics, premature rupture of the membranes (PROM) is a frequent observation which is responsible for many premature deliveries. PROM is also associated with an increased risk of fetal and maternal infections. Early diagnosis is mandatory in order to decrease such complications. Despite that current biological tests allowing the diagnosis of PROM are both sensitive and specific, contamination of the samples by maternal blood can induce false positive results. Therefore, in order to identify new potential markers of PROM (present only in amniotic blood, and absent in maternal blood), proteomic studies were undertaken on samples collected from six women at terms (pairs of maternal plasma and amniotic fluid) as well as on four samples of amniotic fluid collected from other women at the 17(th) week of gestation. All samples (N = 16) were analyzed by two-dimensional (2-D) high-resolution electrophoresis, followed by sensitive silver staining. The gel images were studied using bioinformatic tools. Analyses were focused on regions corresponding to pI between 4.5 and 7 and to molecular masses between 20 and 50 kDa. In this area, 646 +/- 113 spots were detected, and 27 spots appeared to be present on the gels of amniotic fluid, but were absent on those of maternal plasma. Nine out of these 27 spots were also observed on the gels of the four samples of amniotic fluids collected at the 17(th) week of pregnancy. Five of these 9 spots were unambiguously detected on preparative 2-D gels stained by Coomassie blue, and were identified by mass spectrometry analyses. Three spots corresponded to fragments of plasma proteins, and 2 appeared to be fragments of proteins not known to be present in plasma. These 2 proteins were agrin (SWISS-PROT: O00468) and perlecan (SWISS-PROT: P98160). Our results show that proteomics is a valuable approach to identify new potential biological markers for future PROM diagnosis.     

Rapid microtubule-dependent induction of neurite-like extensions in NIH 3T3 fibroblasts by inhibition of ROCK and Cbl

A number of key cellular functions, such as morphological differentiation and cell motility, are closely associated with changes in cytoskeletal dynamics. Many of the principal signaling components involved in actin cytoskeletal dynamics have been identified, and these have been shown to be critically involved in cell motility. In contrast, signaling to microtubules remains relatively uncharacterized, and the importance of signaling pathways in modulation of microtubule dynamics has so far not been established clearly. We report here that the Rho-effector ROCK and the multiadaptor proto-oncoprotein Cbl can profoundly affect the microtubule cytoskeleton. Simultaneous inhibition of these two signaling molecules induces a dramatic rearrangement of the microtubule cytoskeleton into microtubule bundles. The formation of these microtubule bundles, which does not involve signaling by Rac, Cdc42, Crk, phosphatidylinositol 3-kinase, and Abl, is sufficient to induce distinct neurite-like extensions in NIH 3T3 fibroblasts, even in the absence of microfilaments. This novel microtubule-dependent function that promotes neurite-like extensions is not dependent on net changes in microtubule polymerization or stabilization, but rather involves selective elongation and reorganization of microtubules into long bundles.     

Recognition of cyclonucleoside lesions by the Lactococcus lactis FPG protein

Several purine and pyrimidine cyclonucleosides were found to be not recognized by several Escherichia coli and yeast DNA N-glycosylases. Interestingly, a non covalent complex was observed between the Lactoccocus lactis formamidopyrimidine-DNA glycosylases (Fpg-Ll) and the cyclonucleosides. This may provide new information on the mechanism involved in the activity of the latter enzyme.     

Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus

We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures.