Large scale purification of linear plasmid DNA for efficient high throughput cloning

International audienceIn this report we describe a rapid, simple, and efficient method for large scale purification of linear plasmid DNA to answer demand from high throughput gene cloning. The process is based on the separation of the linear vector from small DNA fragments by anion exchange chromatography. Gene cloning experiments by restriction/ligation or the In-Fusion™ technique confirmed the high quality of the linearized vector as 100% of the genes were successfully cloned

Phosphomimetic mutations modulate the ability of HIV-1 Rev to bind human Importin β <em>in vitro</em>

International audienceThe HIV-1 Rev (Regulator of Expression of Virion) protein, an RNA-binding protein essential for viral replication, is imported into the host cell nucleus by human Importin β (Impβ). Rev is phosphorylated in vivo on serine residues by a nuclear kinase. In this study, we introduced glutamate substitution mutations that mimic phosphorylation at serine positions previously identified as potential phosphorylation sites and assessed their impact on the ability of Rev to bind Impβ in thermal shift, gel shift, and fluorescence polarization assays. Phosphomimetic mutations introduced in either the N-terminal tail, helical hairpin domain or C-terminal domain of Rev had a small but reproducible stabilizing effect on the Impβ/Rev complex. Moreover, the mutation of Rev residue Ser56, which localizes to one face of the helical hairpin domain, had a greater stabilizing effect than that of Ser54 located on the opposite face, suggesting that the helical hairpin orients its Ser56-containing face towards Impβ. Taken together, our results suggest that phosphorylation can significantly modulate the ability of Rev to associate with Impβ

Expression and purification of FtsW and RodA from Streptococcus pneumoniae, two membrane proteins involved in cell division and cell growth, respectively

FtsW and RodA are homologous integral membrane proteins involved in bacterial cell division and cell growth, respectively. Both proteins from Streptococcus pneumoniae were overexpressed in Escherichia coli as N-terminal His-tagged fusions. Their membrane addressing in E. coli was demonstrated by cell fractionation and was confirmed for FtsW by immunolocalization. Recombinant FtsW and RodA were solubilized from membranes using 3-(laurylamido)-N,N'-dimethylaminopropylamine oxide (LAPAO). The detergent was exchanged to polyoxyethylene 8 lauryl ether (C12E8) during one-step purification procedure by Co(2+)-affinity chromatography. This procedure yielded 50-150 microg protein per litre of culture. Both proteins are likely to be folded as they are resistant to trypsin digestion and could be incorporated into reconstituted lipid vesicles

IS1 transposition is enhanced by translation errors and by bacterial growth at extreme glucose levels

Transposition of insertion sequences (IS) is an enzyme-mediated process that only occurs in a minority of cells within a bacterial culture. Transposition is thus a rare event, but transposition frequency may vary depending on experimental conditions. For instance in a rich broth, IS elements are known to transpose during stationary phase but not during exponential growth. Using a reporter system which involves the activation of the cryptic bgl operon in Escherichia coli, we show that the frequency of IS1 transposition is a function of glucose concentration in the growth medium, it is increased by streptomycin amounts that are below minimum inhibitory concentration (sub-MIC) and is inhibited in an rpsL150 strain with high translation accuracy. Since starved cells are known to enhance ribosome frameshifting, our data suggests that growth conditions applied in this study could affect IS1 transposition by increasing translation infidelity

Structure of a fluorescent protein from Aequorea victoria bearing the obligate-monomer mutation A206K

The green fluorescent protein (GFP) from the jellyfish Aequoria victoria has been shown to dimerize at high concentrations, which could lead to artefacts in imaging experiments. To ensure a truly monomeric state, an A206K mutation has been introduced into most of its widely used variants, with minimal effect on the spectroscopic properties. Here, the first structure of one of these variants, the cyan fluorescent protein mTurquoise, is presented and compared with that of its dimeric version mTurquoise-K206A. No significant structural change is detected in the chromophore cavity, reinforcing the notion that this mutation is spectroscopically silent and validating that the structural analysis performed on dimeric mutants also applies to monomeric versions. Finally, it is explained why cyan versions of GFP containing the Y66W and N146I mutations do not require the A206K mutation to prevent dimerization at high concentrations

Chromophore Isomer Stabilization Is Critical to the Efficient Fluorescence of Cyan Fluorescent Proteins

International audienceECFP, the first usable cyan fluorescent protein (CFP), was obtained by adapting the tyrosine-based chromophore environment in green fluorescent protein to that of a tryptophan-based one. This first-generation CFP was superseded by the popular Cerulean, CyPet, and SCFP3A that were engineered by rational and random mutagenesis, yet the latter CFPs still exhibit suboptimal properties of pH sensitivity and reversible photobleaching behavior. These flaws were serendipitously corrected in the third-generation CFP mTurquoise and its successors without an obvious rationale. We show here that the evolution process had unexpectedly remodeled the chromophore environment in second-generation CFPs so they would accommodate a different isomer, whose formation is favored by acidic pH or light irradiation and which emits fluorescence much less efficiently. Our results illustrate how fluorescent protein engineering based solely on fluorescence efficiency optimization may affect other photophysical or physicochemical parameters and provide novel insights into the rational evolution of fluorescent proteins with a tryptophan-based chromophore

Reconstitution of membrane protein complexes involved in pneumococcal septal cell wall assembly.

The synthesis of peptidoglycan, the major component of the bacterial cell wall, is essential to cell survival, yet its mechanism remains poorly understood. In the present work, we have isolated several membrane protein complexes consisting of the late division proteins of Streptococcus pneumoniae: DivIB, DivIC, FtsL, PBP2x and FtsW, or subsets thereof. We have co-expressed membrane proteins from S. pneumoniae in Escherichia coli. By combining two successive affinity chromatography steps, we obtained membrane protein complexes with a very good purity. These complexes are functional, as indicated by the retained activity of PBP2x to bind a fluorescent derivative of penicillin and to hydrolyze the substrate analogue S2d. Moreover, we have evidenced the stabilizing role of protein-protein interactions within each complex. This work paves the way for a complete reconstitution of peptidoglycan synthesis in vitro, which will be critical to the elucidation of its intricate regulation mechanisms

In vitro reconstitution of a trimeric complex of DivIB, DivIC and FtsL, and their transient co-localization at the division site in Streptococcus pneumoniae.

International audienceDivIB, DivIC and FtsL are bacterial proteins essential for cell division, which show interdependencies for their stabilities and localization. We have reconstituted in vitro a trimeric complex consisting of the recombinant extracellular domains of the three proteins from Streptococcus pneumoniae. The extracellular domain of DivIB was found to associate with a heterodimer of those of DivIC and FtsL. The heterodimerization of DivIC and FtsL was artificially constrained by fusion with interacting coiled-coils. Immunofluorescence experiments showed that DivIC is always localized at mid-cell, in contrast to DivIB and FtsL, which are co-localized with DivIC only during septation. Taken together, our data suggest that assembly of the trimeric complex DivIB/DivIC/FtsL is regulated during the cell cycle through controlled formation of the DivIC/FtsL heterodimer