Yann Chevolot

MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms

BackgroundThe production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms.ResultsWe present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented.ConclusionsThe objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation.

Specific recognition of lectins by oligonucleotide glycoconjugates and sorting on a DNA microarray.

Two glycoconjugates bearing different DNA tags are mixed in solution with lectins; both interact with their specific lectin and the resulting complexes are sorted, according to their DNA sequences, at the surface of micro-reactors bearing the immobilised complementary DNA sequences.

Engineering and Characterization of Polymer Surfaces for Biomedical Applications

The application of synthetic polymers in the growing field of materials for medical applications is illustrated by examples from recent work at the Materials Institute of the Swiss Federal Institute of Technology in Lausanne. The review highlights the need for functionalization and chemical control of material surfaces at a molecular/functional level. After a brief introduction into the surface chemical analysis tools, i.e., X-ray Photoelectron Spectroscopy (XPS) and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) combined with contact angle measurements, phosphorylcholine biomimicking polymers as well as immobilization of carbohydrates on polystyrene are presented.

New concepts of integrated photonic biosensors based on porous silicon

A biosensor is a device that uses specific biochemical reactions mediated by isolated tissues, enzymes, immunosystems, organelles or whole cells to detect chemical compounds (IUPAC: http://goldbook.iupac.org/B00663.html). Biosensors integrate two functions, i) a bioreceptor functionalized with probes able to specifically recognize the targeted species and ii) a transducer converting the specific biological interaction into a quantitatively measurable signal. One way to classify biosensors relates on the transduction mode, such as optical (fluorescence, surface-enhanced Raman scattering, chemiluminescence, colorimetry, dual polarization interferometry and surface plasmon resonance), electrochemical (amperometry, potentiometry, field-effect transistor and conductimetry) and gravimetric transduction (quartz crystal microbalance, cantilever) (Sassolas et al., 2008). The evaluation of biosensor performances relies on the following criteria: high sensitivity, operational and linear concentration range, detection and quantitative determination limits, high selectivity, steady-state and transient response times, sample throughput, reliability, reproducibility, stability and long lifetime (Thevenot et al., 1999). Other aspects like cost of test, ease of use, time of analysis including all the steps required for sample preparation should also be taken into account. Some biosensors are based on the use of labels such as colorimetric, fluorescent, enzymatic moieties or redox species... However, the current trends aim to develop on-chip integrated and label free detection systems. In this framework, porous silicon (PSi) offers high potential for biosensing: • PSi physical properties directly depend on the structure. The optical properties are linked to the variation of refractive index with a change of porosity while the electrochemical properties rely on surface chemistry modification. Thus, PSi based transducers can be sensitive both to surface or volume biomolecular recognition. • PSi surface chemistry is essentially governed by the high reactivity of Si-H bond, which can form both Si-alkyle or Si-OH bond (Stewart & Buriak, 2000). Thus, the surface can be either hydrophobic or hydrophilic, and a large range of biomolecules can be immobilized.

Acid deprotection of covalently immobilized peptide probes on glass slides for peptide microarrays

Protein microarray technology has shown great advancements in the field of biomedical research and diagnosis, it allows to study and understand protein activities and protein-ligand interactions (e.g. detection of antigen-autoantibody interaction in autoimmune diseases. Autoantibodies are frequently targeted against antigens of the cell nucleus (double and single stranded DNA, histones, and nuclear antigens). The biological activities of proteins (e.g. enzymes, antibodies...) are controlled by peptides sequences of the active site. Consequently, we were interested in the investigation of peptide microarrays in order to further implement in situ peptide synthesis, in particular, deprotection reaction on glass supported peptides. In this work, a protected and biotinylated synthetic peptide was covalently immobilized onto amino functionalized glass surface by activation of its the C-terminus; this allows to orientate the peptide onto the surface. The peptide contains a fragment of the C-terminal end of the human histone H3 protein. The immobilized peptide was then deprotected by using concentrated trifluoroacetic acid solution. After the deprotection, surface stability and peptide grafting density were evaluated by indirect labelling of the immobilized peptide using Cy3 streptavidin conjugates. We also studied biological interaction of IgG polyclonal anti-histone H3 antibody with the immobilized peptide epitope to insure the efficiency of the acid deprotection. The specificity of the antibody interaction with the protected versus non protected peptides. This approach may be applied to in situ synthetic and prototected peptides, in order to elaborate a micro-immunoassay prototype for measurement of peptide-protein interactions on high density microarrays, and detection of antibodies in biological fluids such as serum.

PREPARATION OF CORE-SHELL SILVER/SILICA NANOPATICLES AND THEIR APPLICATION FOR ENHANCEMENT OF CYANINE 3 FLUORESCENCE

Core shell Ag@SiO2-Streptavidin-Cy3 nanoparticles were prepared. Ag@SiO2 nanoparticles were synthesized via a sol–gel method. Then, Streptavidin-Cy3 was covalently bonded to the Ag@SiO2 surface. These core-shell nanoparticles were characterized by steady-state fluorescence spectroscopy and fluorescence scanning. In presence of the silver core, a 2.5-time enhancement of Cy3 fluorescence intensity was obtained. This result shows that these nanoparticles can be potentially helpful in surface analysis based on biochip.

Carbohydrates as Recognition Receptors in Biosensing Applications

Carbohydrates are involved in crucial physiological and pathological events. One can take advantage of carbohydrate-based interaction for drug discovery, diagnosis, antibiotics, vaccine, etc. This chapter deals with biosensors and microarrays that take advantage of carbohydrates-based interactions with a special interest in devices that are designed for medical applications. A large overview of glycochemistry, followed by the biological role of carbohydrates, is given. Carbohydrate-based biosensors are then described with special emphasis on surface chemistry and signal transduction. Finally, medically relevant applications illustrate the use of carbohydrates as recognition receptors in biosensing.

SELF-ASSEMBLY ARCHITECTURES OF NEW DNA-BASED STRUCTURES IN AIR AND IN LIQUIDS ANALYZED BY ATOMIC FORCE MICROSCOPY

The architecture of self assembled X shaped DNA structure was studied by AFM on mica. The dimensions and extent of self assembled structures were influenced by mica surface treatment and depended on whether observations were performed in air or in Tris buffer solution. A molecular model is proposed.

ELABORATION AND GRAFTING OF MAGNETIC BEAD-CHAINS FOR DETECTION OF ANISOTROPY WITH POLARIMETRIC SURFACE PLASMON RESONANCE IMAGING SYSTEM

The aim of this report is to prove that polarimetric surface plasmon resonance imaging (P-SPRI) is able to characterize dynamically the anisotropy of micro and nanoobjects. Micro and nanoparticles were assembled into filaments at a specific location on a support, using a combination of magnetic field, amine based chemistry and orthogonal surface chemistry. After immobilization of the filaments onto gold pads, they were actuated by a magnetic field and validated the P-SPRI system.

Integrated Microfluidic-Microoptical Detection Systems Fabricated by Dry Etching of Soda-Lime Glass

In this paper, a method of fabrication of a microfluidic system, integrated with fluorescence detection optics, is presented. Soda-lime glass is used as a substrate material, in which the waveguiding optics is fabricated by means of an ion exchange process, whereas the microfluidic channel is manufactured utilising reactive ion etching technique. Basic optical characteristics of the device are also reported.Copyright