13 research outputs found
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
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
Advances in heterologous expression of recombinant proteins
No full textProtein production for structural and biophysical studies, functional assays, biomarkers, mechanistic studies in vitro and in vivo, but also for therapeutic applications in Pharma, Biotech and Academia has evolved into a mature discipline in recent years. Due to the increased emphasis on biopharmaceuticals, the growing demand for proteins used for structural and biophysical studies, the impact of genomics technologies on the analysis of large sets of structurally diverse proteins, and the increasing complexity of disease targets the interest in innovative approaches for the expression, purification and characterisation of recombinant proteins has steadily increased over the years.
In this review, we summarise recent developments in the field of recombinant protein expression for research use in Pharma, Biotech and Academia. We focus mostly on the latest developments for protein expression in the most widely used expression systems: Escherichia coli (E. coli), insect cell expression using the Baculovirus Expression Vector System (BEVS) and, finally, transient and stable expression of recombinant proteins in mammalian cells
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
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 ()
() Expression of viral and human protein/protein domains in Rosetta(DE3)LysS
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> Plasmids were transformed into Rosetta(DE3)pLysS cells, expression was auto-induced in TB Overnight Express™ and the resulting expression levels assayed by a Ni-NTA robotic screen, followed by SDS-PAGE. The lane labels refer to unique OPPF identifiers (OPPF construct numbers; see Supplementary Table 2) followed by the pOPIN vector name (pOPINF contributes an N-His-3C site fusion to the protein of interest, pOPINJ contributes an N-His-GST-3C site fusion to the protein of interest and pOPINM contributes an N-His-MBP-3C site fusion to the protein of interest). eGFP refers to enhanced GFP and ‘null’ refers to vector alone (i.e. vector with no target genes inserted), pOPINJ and pOPINM will however express N-His-GST (29.5 kDa) and N-His-MBP (44.3 kDa), respectively. The lane labelled ‘Low’ contains Low Range Sigma Markers (M3913) and the lane labelled ‘High’ contains High Range Sigma Markers (M3788), molecular marker masses are indicated in kDa. () Expression of viral and human protein/protein domains in HEK293T cells. Plasmids were robotically transfected into HEK293T cells and the resulting expression levels assayed by a SDS-PAGE and western blotting as described in the Materials and methods section. The lane labels are identical to panel A except that the standard molecular weight markers are replaced with marked positions of the BenchMark™ ladder (InVitrogen 10747-012), His-tagged molecular mass markers (masses in kDa) on the right. () Expression of viral and human protein/protein domains in 9 cells. Plasmids were robotically co-transfected into HEK293T cells with linearized bacmid and the resulting expression levels assayed by a SDS-PAGE and western blotting as described in the Materials and methods section. The lane labels are identical to panel A except that the standard molecular weight markers are replaced with marked positions of the BenchMark™ ladder (InVitrogen 10747-012), His-tagged molecular mass markers (masses in kDa) on the right
Structure of the Murray Valley encephalitis virus RNA helicase at 1.9 Angstrom resolution.
No full textMurray Valley encephalitis virus (MVEV), a mosquito-borne flavivirus endemic to Australia, is closely related to Japanese encephalitis virus and West Nile virus. Nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains, whose activity is central to flavivirus replication and is therefore a possible target for anti-flaviviral compounds. Cloning, purification, and crystal structure determination to 1.9 Angstrom resolution of the NS3 helicase of MVEV and characterization of its enzymatic activity is reported. Comparison with the structures of helicases from related viruses supports a possible mechanism of ATP hydrolysis-driven strand separation
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
Lysine methylation as a routine rescue strategy for protein crystallization.
No full textCrystallization remains a critical step in X-ray structure determination. Because it is not generally possible to rationally predict crystallization conditions, commercial screens have been developed which sample a wide range of crystallization space. While this approach has proved successful in many cases, a significant number of proteins fail to crystallize despite being soluble and monodispersed. It is established that chemical modification can facilitate the crystallization of otherwise intractable proteins. Here we describe a method for the reductive methylation of lysine residues which is simple, inexpensive, and efficient, and report on its application to ten proteins. We describe the effect of methylation on the physico-chemical properties of these proteins, and show that it led to diffraction-quality crystals from four proteins and structures for three that had hitherto proved refractory to crystallization. The method is suited to both low- and high-throughput laboratories
