44 research outputs found
The electrophoretic mobility of DNA three-way junctions is affected by the sequence of overhanging single-stranded ends
No full textThe folding of three- and four-way DNA junctions is often assessed by comparing the electrophoretic mobility of restriction enzyme fragments, using the long-short arm assay. We have compared the mobility of synthetic three-way junctions that contain identical branch point sequences, but different restriction sites in the arms. We show that the mobility of fragments is affected by the sequence of the overhanging ends. In general, GC-rich overhangs produce fragments with anomalous mobilities. These anomalies can be prevented by treating the cleaved junctions with mung bean endonuclease, elevating the electrophoresis temperature or using blunt cleaving restriction endonuclease
Sequence-dependent folding of DNA three-way junctions
No full textThree-way DNA junctions can adopt several different conformers, which differ in the coaxial stacking of the arms. These structural variants are often dominated by one conformer, which is determined by the DNA sequence. In this study we have compared several three-way DNA junctions in order to assess how the arrangement of bases around the branch point affects the conformer distribution. The results show that rearranging the different arms, while retaining their base sequences, can affect the conformer distribution. In some instances this generates a structure that appears to contain parallel coaxially stacked helices rather than the usual anti-parallel arrangement. Although the conformer equilibrium can be affected by the order of purines and pyrimidines around the branch point, this is not sufficient to predict the conformer distribution. We find that the folding of three-way junctions can be separated into two groups of dinucleotide steps. These two groups show distinctive stacking properties in B-DNA, suggesting there is a correlation between B-DNA stacking and coaxial stacking in DNA junctions
Introduction to Protein Production - Current challenges in protein science: from fundamental research to drug development
No full textIntroduction to Protein Production - Current challenges in protein science: from fundamental research to drug developmen
Groundwater and solute transport modelling study Vosdonk Noord at Etten-Leur: Examining the effect of two implementation methodologies for highly heterogenic shallow subsurface characteristics
No full textThe industrial site of Vosdonk Noord at Etten-Leur in the Netherlands consists of a large soil contamination in combination with highly heterogenic shallow subsurface soil characteristics. In this report, we study the groundwater flow and solute transport behaviour at this project location. Throughout this process, knowledge is gathered about the interpretation of the shallow subsurface heterogeneity with a main focus on the hydraulic conductivities. It is interesting to look at the subsurface heterogeneity because of the challenge to implement it inside a model and its uncertainty in characteristics. This means the subsurface heterogeneity is part of the problem to be solved. A comparison of groundwater flow and solute transport results were made using kriging as an interpolation method to implement subsurface cone penetration test data directly into the model. This generated a cell by cell implementation of the subsurface characteristics. To include the possible variability of the subsurface and to increase the reliability of the results, random simulations were implemented. In practice, the “pancake” method characterises the subsurface in a commercial software like Visual Modflow. This “pancake” method uses continuous horizontal subsurface soil layers. The gathered knowledge is useful to try and tackle the in practice used “pancake” method in case of a highly heterogenic subsurface
The structure of the nucleoprotein binding domain of lyssavirus phosphoprotein reveals a structural relationship between the N-RNA binding domains of Rhabdoviridae and Paramyxoviridae.
No full textThe phosphoprotein P of non-segmented negative-sense RNA viruses is an essential component of the replication and transcription complex and acts as a co-factor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. We have obtained the structure of the C-terminal domain of P of Mokola virus (MOKV), a lyssavirus that belongs to the Rhabdoviridae family and mapped at the amino acid level the crucial positions involved in interaction with N and in the formation of the viral replication complex. Comparison of the N-RNA binding domains of P solved to date suggests that the N-RNA binding domains are structurally conserved among paramyxoviruses and rhabdoviruses in spite of low sequence conservation. We also review the numerous other functions of this domain and more generally of the phosphoprotein
Structure and functionality in flavivirus NS-proteins: Perspectives for drug design
No full textFlaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads
Recent advances in the production of proteins in insect and mammalian cells for structural biology.
No full textThe production of proteins in sufficient quantity and of appropriate quality is an essential pre-requisite for structural studies. Escherichia coli remains the dominant expression system in structural biology with nearly 90% of the structures in the Protein Data Bank (PDB) derived from proteins produced in this bacterial host. However, many mammalian and eukaryotic viral proteins require post-translation modification for proper folding and/or are part of large multimeric complexes. Therefore expression in higher eukaryotic cell lines from both invertebrate and vertebrate is required to produce these proteins. Although these systems are generally more time-consuming and expensive to use than bacteria, there have been improvements in technology that have streamlined the processes involved. For example, the use of multi-host vectors, i.e., containing promoters for not only E. coli but also mammalian and baculovirus expression in insect cells, enables target genes to be evaluated in both bacterial and higher eukaryotic hosts from a single vector. Culturing cells in micro-plate format allows screening of large numbers of vectors in parallel and is amenable to automation. The development of large-scale transient expression in mammalian cells offers a way of rapidly producing proteins with relatively high throughput. Strategies for selenomethionine-labelling (important for obtaining phase information in crystallography) and controlling glycosylation (important for reducing the chemical heterogeneity of glycoproteins) have also been reported for higher eukaryotic cell expression systems
Automation of large scale transient protein expression in mammalian cells
No full textTraditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI⁻ cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative
Expression of KIAA0319 proteins in HEK 293T cells using the pOPING vector
No full text<p><b>Copyright information:</b></p><p>Taken from "A versatile ligation-independent cloning method suitable for high-throughput expression screening applications"</p><p></p><p>Nucleic Acids Research 2007;35(6):e45-e45.</p><p>Published online 22 Feb 2007</p><p>PMCID:PMC1874605.</p><p>© 2007 The Author(s)</p> Domain and multi-domain constructs were screened for expression in HEK293T cells and subsequent secretion into the cell media analysed using SDS-PAGE and western blotting as described in the Materials and methods section. The lane labels refer to unique OPPF identifiers (OPPF contruct numbers: see Supplementary Table 1). The lane labelled BM contains the BenchMark™ ladder (InVitrogen 10747-012), molecular marker masses are indicated in kDa. Construct numbers labelled * were selected for scale-up and purification ()
