A thermo-responsive, self-assembling biointerface for on demand release of surface-immobilised proteins

Dedicated chemistries for on-demand capture and release of biomolecules at the solid-liquid interface are required for applications in drug delivery, for the synthesis of switchable surfaces used in analytical devices and for the assembly of next-generation biomaterials with complex architectures and functions. Here we report the engineering of a binary self-assembling polypeptide system for reversible protein capture, immobilisation and controlled thermo-responsive release from a solid surface. The first element of the binary system is a universal protein substrate immobilised on a solid surface. This protein is bio-inspired by the neuronal SNAP25, which is the protein involved in the docking and fusion of synaptic vesicles to the synaptic membrane. The second element is an artificial chimeric protein engineered to include distinct domains from three different proteins: Syntaxin, VAMP and SNAP25. These native proteins constitute the machinery dedicated to vesicle trafficking in eukaryotes. We removed approximately 70% of native protein sequence from these proteins and constructed a protein chimera capable of high affinity interaction and self-assembly with immobilised substrate. The interaction of the two parts of the engineered protein complex is strong but fully-reversible and therefore the chimera can be recombinantly fused as a tag to a protein of interest, to allow spontaneous assembly and stimuli-sensitive release from the surface upon heating at a predetermined temperature. Two thermo-responsive tags are reported: the first presents remarkable thermal stability with melting temperature of the order of 80°C; the second disassembles at a substantially lower temperature of about 45°C. The latter is a promising candidate for remote-controlled localised delivery of therapeutic proteins, as physiologically tolerable local increase of temperatures in the 40-45°C range can be achieved using magnetic fields, infra-red light or focused ultrasound. Importantly, these two novel polypeptides provide a broader blueprint for the engineering of future functional proteins with predictable folding and response to external stimuli

The model of specialists training system for high-tech industries

The article reviews the world experience of the specialist training model for high-tech industries. The focus is made on the approach to organization of training to help students develop enterprise-technological skills implemented in National Research Tomsk Polytechnic University. The model considers the strategy of scientific and technological development adopted in the Russian Federation and the need of high-tech industries to improve the quality of educational and training programs and training of the personnel to meet the demand of today's environment who are ready to face future challenges and to establish new markets, and creates the conditions for development of entrepreneurial and technological competences

Labelling of live cells using fluorescent aptamers: binding reversal with DNA nucleases

A reversible cell labelling method has been developed for non-destructive and non-invasive cell labelling and purification. Our method uses high affinity single strand DNA (ssDNA) aptamers against surface exposed target molecules on cells. The aptamers are subsequently removed from the cell surface using DNase nuclease treatment. We exemplified our method by labelling human acute lymphoblastic leukemia cells with Qdot-ssDNA aptamers, and restoring them to the label-free condition by treatment with Benzonase. Binding of the fluorescent-aptamers to the cells was evaluated by measuring fluorescence intensity and was further confirmed using flow cytometry. Removal of the aptamers can be achieved in ~10 min by the DNase nuclease digestion. Incubation of cells with aptamers or with the nucleases results in no apparent damage to the cells and does not affect their growth rates. The latter were equivalent to the rates measured for the untreated cells. Our method provides an alternative to traditional antibody-based techniques and could be especially suitable for non-invasive reversible cell labelling and cell separations where maintaining native cell activity is needed

Selection strategy and the design of hybrid oligonucleotide primers for RACE-PCR: cloning a family of toxin-like sequences from Agelena orientalis

<p>Abstract</p> <p>Background</p> <p>the use of specific but partially degenerate primers for nucleic acid hybridisations and PCRs amplification of known or unknown gene families was first reported well over a decade ago and the technique has been used widely since then.</p> <p>Results</p> <p>here we report a novel and successful selection strategy for the design of hybrid partially degenerate primers for use with RT-PCR and RACE-PCR for the identification of unknown gene families. The technique (named PaBaLiS) has proven very effective as it allowed us to identify and clone a large group of mRNAs encoding neurotoxin-like polypeptide pools from the venom of <it>Agelena orientalis </it>species of spider. Our approach differs radically from the generally accepted CODEHOP principle first reported in 1998. Most importantly, our method has proven very efficient by performing better than an independently generated high throughput EST cloning programme. Our method yielded nearly 130 non-identical sequences from <it>Agelena orientalis</it>, whilst the EST cloning technique yielded only 48 non-identical sequences from 2100 clones obtained from the same <it>Agelena </it>material. In addition to the primer design approach reported here, which is almost universally applicable to any PCR cloning application, our results also indicate that venom of <it>Agelena orientalis </it>spider contains a much larger family of related toxin-like sequences than previously thought.</p> <p>Conclusion</p> <p>with upwards of 100,000 species of spider thought to exist, and a propensity for producing diverse peptide pools, many more peptides of pharmacological importance await discovery. We envisage that some of these peptides and their recombinant derivatives will provide a new range of tools for neuroscience research and could also facilitate the development of a new generation of analgesic drugs and insecticides.</p

Self-assembly of proteins and their nucleic acids

We have developed an artificial protein scaffold, herewith called a protein vector, which allows linking of an in-vitro synthesised protein to the nucleic acid which encodes it through the process of self-assembly. This protein vector enables the direct physical linkage between a functional protein and its genetic code. The principle is demonstrated using a streptavidin-based protein vector (SAPV) as both a nucleic acid binding pocket and a protein display system. We have shown that functional proteins or protein domains can be produced in vitro and physically linked to their DNA in a single enzymatic reaction. Such self-assembled protein-DNA complexes can be used for protein cloning, the cloning of protein affinity reagents or for the production of proteins which self-assemble on a variety of solid supports. Self-assembly can be utilised for making libraries of protein-DNA complexes or for labelling the protein part of such a complex to a high specific activity by labelling the nucleic acid associated with the protein. In summary, self-assembly offers an opportunity to quickly generate cheap protein affinity reagents, which can also be efficiently labelled, for use in traditional affinity assays or for protein arrays instead of conventional antibodies

Quantum dots improve peptide detection in MALDI MS in a size dependent manner

Laser Desorption Ionization Mass Spectrometry employs matrix which is co-crystallised with the analyte to achieve "soft ionization" that is the formation of ions without fragmentation. A variety of matrix-free and matrix-assisted LDI techniques and matrices have been reported to date. LDI has been achieved using ultra fine metal powders (UFMPs), desorption ionisation on silicon (DIOS), sol-gel assisted laser desorption/ionization (SGALDI), as well as with common MALDI matrices such as 2,5-dihydroxy benzoic acid (DHB), 3,5-dimethoxy-4-hydroxycinnamic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA) to name a few. A variety of matrix additives have been shown to improve matrix assisted desorption, including silicon nanowires (SiNW), carbon nanotubes (CNT), metal nanoparticles and nanodots. To our knowledge no evidence exists for the application of highly fluorescent CdSe/ZnS quantum dots to enhance MALDI desorption of biological samples. Here we report that although CdSe/ZnS quantum dots on their own can not substitute matrix in MALDI-MS, their presence has a moderately positive effect on MALDI desorption, improves the signal-to-noise ratio, peak quality and increases the number of detected peptides and the overall sequence coverage

Combinatorial peptidomics: a generic approach for protein expression profiling

Traditional approaches to protein profiling were built around the concept of investigating one protein at a time and have long since reached their limits of throughput. Here we present a completely new approach for comprehensive compositional analysis of complex protein mixtures, capable of overcoming the deficiencies of current proteomics techniques. The Combinatorial methodology utilises the peptidomics approach, in which protein samples are proteolytically digested using one or a combination of proteases prior to any assay being carried out. The second fundamental principle is the combinatorial depletion of the crude protein digest (i.e. of the peptide pool) by chemical crosslinking through amino acid side chains. Our approach relies on the chemical reactivities of the amino acids and therefore the amino acid content of the peptides (i.e. their information content) rather than their physical properties. Combinatorial peptidomics does not use affinity reagents and relies on neither chromatography nor electrophoretic separation techniques. It is the first generic methodology applicable to protein expression profiling, that is independent of the physical properties of proteins and does not require any prior knowledge of the proteins. Alternatively, a specific combinatorial strategy may be designed to analyse a particular known protein on the basis of that protein sequence alone or, in the absence of reliable protein sequence, even the predicted amino acid translation of an EST sequence. Combinatorial peptidomics is especially suitable for use with high throughput micro- and nano-fluidic platforms capable of running multiple depletion reactions in a single disposable chip

Using single cell cultivation system for on-chip monitoring of the interdivision timer in Chlamydomonas reinhardtii cell cycle

Regulation of cell cycle progression in changing environments is vital for cell survival and maintenance, and different regulation mechanisms based on cell size and cell cycle time have been proposed. To determine the mechanism of cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii, we developed an on-chip single-cell cultivation system that allows for the strict control of the extracellular environment. We divided the Chlamydomonas cell cycle into interdivision and division phases on the basis of changes in cell size and found that, regardless of the amount of photosynthetically active radiation (PAR) and the extent of illumination, the length of the interdivision phase was inversely proportional to the rate of increase of cell volume. Their product remains constant indicating the existence of an 'interdivision timer'. The length of the division phase, in contrast, remained nearly constant. Cells cultivated under light-dark-light conditions did not divide unless they had grown to twice their initial volume during the first light period. This indicates the existence of a 'commitment sizer'. The ratio of the cell volume at the beginning of the division phase to the initial cell volume determined the number of daughter cells, indicating the existence of a 'mitotic sizer'