Biomolecular engineering最新文献

Mutations in the dystrophin gene result in both Duchenne and Becher muscular dystrophies (DMD and BMD). Approximately 65% of all mutations causing DMD are deletions (60%) or duplications (5%) of large segments of this gene, spanning one exon or more. Due to the large size of the dystrophin gene (79 exons), finding point mutations has been prohibitively expensive and laborious. Recent studies confirm the utility of pre-screening methods, as denaturing high-performance liquid chromatography (DHPLC) analysis in the identification of point mutations in the dystrophin gene, with an increment of mutation detection rate from 65% to more than 92%. Here we suggest an alternative and convenient method of DHPLC analysis in order to find mutations in a more rapid and less expensive way by introducing the analysis of 16 couples of dystrophin amplicons, in biplex exons DHPLC runs. Using this new protocol of biplex exons DHPLC screening, new mutations were identified in four male patients affected by DMD who had tested negative for large DNA rearrangements.

Antibody engineering has become a well-developed discipline, encompassing discovery methods, production strategies, and modification techniques that have brought forth clinically investigated and marketed therapeutics. The realization of the long-standing goal of production of fully human monoclonal antibodies has focused intensive research on the clinical employment of this potent drug category. However, antibodies are large macromolecules that pose numerous challenges in formulation, optimal pharmacokinetics, manufacturing, stability, and process development. While further improvements in discovery technologies, such as phage display, ribosome display, and transgenic animals continue to advance our capacity to rapidly screen and refine optimal binding molecules, antibody engineers have recently focused more of their efforts on improving protein production and stability, as well as engineering improved biological properties in the effector domains of monoclonal antibodies. A second long-standing goal of antibody engineering, the development of targeted drugs, has not been wholly realized, but this obvious application for antibodies is currently undergoing increasing exploration. Minimal binding proteins, such as Fab, scFv, and single variable domains are the preferred targeting elements for some investigational drugs, whereas non-immunoglobulin scaffold proteins have been explored as binding proteins in other designs. The necessity to utilize non-protein components in targeted drugs, such as polymers, linkers, and cytotoxics, has brought a convergence of the fields of bioconjugate chemistry and protein engineering in experimental antibody therapeutics.

Many phytopathogenic species of the fungus Cercospora produce cercosporin, a photoactivated perylenequinone toxin that belongs to a family of photosensitizers, which absorb light energy and produce extremely cytotoxic, reactive oxygen species. The cpd1 (cercosporin photosensitizer detoxification) gene of yeast (Saccharomyces cerevisiae), which encodes for a novel protein with significant similarity to the FAD-dependent pyridine nucleotide reductases, confers resistance to cercosporin when over-expressed in yeast. The aim of this work was to investigate the potential ability of cpd1 gene to confer resistance to cercosporin when expressed in tobacco plants (Nicotiana tabacum). Transgenic tobacco plants were produced using Agrobacterium tumefaciens, with cpd1 integrated as the gene of interest. We report here that expression of cpd1 gene in tobacco can mediate resistance to cercosporin. The involvement of cpd1 gene in the detoxification of the cercosporin reinforces previous observations, which suggested that resistance to cercosporin is mediated by a mechanism involving toxin reduction.

Systematic Evolution of Ligands by EXponential enrichment (SELEX) is an experimental procedure that allows extraction, from an initially random pool of oligonucleotides, of the oligomers with a desired binding affinity for a given molecular target. The procedure can be used to infer the strongest binders for a given DNA or RNA binding protein, and the highest affinity binding sequences isolated through SELEX can have numerous research, diagnostic and therapeutic applications. Recently, important new modifications of the SELEX protocol have been proposed. In particular, a modification of the standard SELEX procedure allows generating a dataset from which protein–DNA interaction parameters can be determined with unprecedented accuracy. Another variant of SELEX allows investigating interactions of a protein with nucleic-acid fragments derived from the entire genome of an organism. We review here different SELEX-based methods, with particular emphasis on the experimental design and on the applications aimed at inferring protein–DNA interactions. In addition to the experimental issues, we also review relevant methods of data analysis, as well as theoretical modeling of SELEX.

The present study utilizes expert neural networks for the prediction of proteins secondary structure. We use three independent networks, one for each structure (alpha, beta and coil) as the first-level processing unit; decision upon the chosen structure for each residue is carried out by a second-level, post-processing unit, which utilizes the Chou and Fasman frequency values F_α and F_β in order to strengthen and/or deplete the probability of the specific structure under investigation. The highest prediction case was 76%.

Our method requires primitive computational means and a relatively small training set, while still been comparable to previous work. It is not meant to be an alternative to the determination of secondary structure by means of free energy minimization, integration of dynamic equations of motion or crystallography, which are expensive, time-consuming and complicated, but to provide additional constrains, which might be considered and incorporated into larger computing setups in order to reduce the initial search space for the above methods.

A novel recombinant Drosophila melanogaster acetylcholinesterase (R-DmAChE) produced in Pichia pastoris was first reported in this study. We cloned the DmAChE cDNA by reverse transcription PCR with removal of the signal for glycosylphosphatidylinositol (GPI) anchor attachment and the endogenous signal peptide coding sequence, and inserted it into P. pastoris vector pPIC9K under control of the alcohol oxidase gene AOX1 promoter (5′ AOX1). The expression cassette of AChE cDNA was then introduced into methylotrophic yeast GS115 and several recombinant strains expressing R-DmAChE were obtained. The secreted R-DmAChE showed high stability in neutral phosphate buffer at 4 °C, and its kinetic parameters were identical to those of the native DmAChE. The bimolecular rate constants of R-DmAChE to dichlorvos, aldicarb and carbaryl were ranging from three to six times higher than of native DmAChE. Within six insecticides, the R-DmAChE was more sensitive than EeAChE, NbAChE and HuAChE. For 10 widely used insecticides, the IC₅₀ values to the R-DmAChE were much lower than those to AChEs commonly used in China. With the R-DmAChE-based assay, samples spiked with three concentrations of pesticides caused enzymatic activity inhibition with R.S.D. of 0–13.7%. These results suggest that the R-DmAChE can be useful for detection of organophosphate and carbamate insecticide residues.

Polyclonal antibody bound Sepharose 4B support has been exploited for the immobilization of bitter gourd peroxidase directly from ammonium sulphate precipitated proteins. Immunoaffinity immobilized bitter gourd peroxidase exhibited high yield of immobilization. IgG-Sepharose 4B bound bitter gourd peroxidase showed a higher stability against heat, chaotropic agents (urea and guanidinium chloride), detergents (cetyl trimethyl ammonium bromide and Surf Excel), proteolytic enzyme (trypsin) and water-miscible organic solvents (propanol, THF and dioxane). The activity of immobilized bitter gourd peroxidase was significantly enhanced in the presence of cetyl trimethyl ammonium bromide and after treatment with trypsin as compared to soluble enzyme.

The aim of this work was to detect the formation of hydrogen peroxide and hydroxyl radicals after ultrasound (US) exposure and test the hypothesis that reactive oxygen species induced by ultrasound can contribute to DNA damage. Formation of reactive oxygen species was observed in incubated medium after sonication with 1 MHz continuous ultrasound at the intensities of 0.61–2.44 W/cm². Free radicals and hydrogen peroxide produced by ultrasound exposure of cells can lead to DNA damage. Comet assay was used to assess the effect of ultrasound on the level of nuclear DNA damage. The nucleated erythrocytes from fish were exposed in vitro to ultrasound at the same intensities and frequency. It was noticed that ultrasound in all used intensities induced DNA damage. The effect was not eliminated by the addition of catalase, which indicates that DNA damage was not caused by hydrogen peroxide only. The results showed that the DNA damage can be repair and this mechanism was the most effective after 30 and 60 min after sonication. Furthermore, the ultrasound-induced DNA damage in the presence of sonosensitizer (Zn- and AlCl-phthalocyanine) was studied. It was noticed that phthalocyaniens (Pcs) alone or with ultrasound did not induce significant changes in the level of DNA damage.

With the completion of the human genome project (HUGO) during recent years, gene function, protein abundance and expression patterns in tissues and cell types have emerged as central areas for the scientific community. A mapped human proteome will extend the value of the genome sequence and large-scale efforts aiming at elucidating protein localization, abundance and function are invaluable for biomarker and drug discovery. This research area, termed proteomics, is more demanding than any genome sequencing effort and to perform this on a wide scale is a highly diverse task. Therefore, the proteomics field employs a range of methods to examine different aspects of proteomics including protein localization, protein–protein interactions, posttranslational modifications and alteration of protein composition (e.g. differential expression) in tissues and body fluids. Here, some of the most commonly used methods, including chromatographic separations together with mass spectrometry and a number of affinity proteomics concepts are discussed and exemplified.

Posttranslational modifications of proteins possess key functions in the regulation of various cellular processes. While they facilitate fast, location-specific and transient reactions to changing conditions in the first place they enhance the already high complexity of a cellular proteome by orders of magnitude. Furthermore, they can utterly alter the properties of the modified protein, thus making a timely analysis even more difficult. While several standardized methods for the analysis of protein phosphorylation and glycosylation have been established most other modifications require tailor-made solutions for a comprehensive analysis. Therefore, we will provide guidelines for the analysis of some important posttranslational modifications that are underrepresented in contemporary literature.