A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease

Dengue virus threatens around 2.5 billion people worldwide; about 50 million become infected every year, and yet no vaccine or drug is available for prevention and/or treatment. The flaviviral NS2B-NS3pro complex is indispensable for flaviviral replication and is considered to be an important drug target. The aim of this study was to develop a simple and generally applicable experimental strategy to construct, purify, and assay a highly active recombinant NS2B(H)-NS3pro complex that would be useful for high-throughput screening of potential inhibitors. The sequence of NS2B(H)-NS3pro was generated by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro complex was expressed in E. coli predominantly as insoluble protein and purified to >95% purity by single-step immobilized metal affinity chromatography. SDS-PAGE followed by immunoblotting of the purified enzyme demonstrated the presence of the NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 37, 21, and 10 kDa bands, respectively. Kinetic parameters, K _m, k _cat, and k _cat/K _m for the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters K _m, k _cat, and k _cat/K _m for Ac-nKRR-amc substrate were 100 μM, 0.112 s^?1, and 1120 M^?1·s^?1, respectively. A simplified procedure for the cloning, overexpression, and purification of the NS2B(H)-NS3pro complex was applied, and a highly active recombinant NS2B(H)-NS3pro complex was obtained that could be useful for the design of high-throughput assays aimed at flaviviral inhibitor discovery.     

Molecular docking of fisetin with AD associated AChE,ABAD and BACE1 proteins

Alzheimer׳s disease (AD) is one of the most common dementias showing slow progressive cognitive decline. Progression of intracerebral accumulation of beta amyloid (Aβ) peptides by the action of amyloid binding alcohol dehydrogenase (ABAD), a mitochondrial enzyme and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and the degradation of Acetylcholinesterase (AChE) the main pathological characteristics of AD. Therefore, it is of interest to evaluate the importance of fisetin (a flavonol that belongs to the flavonoid group of polyphenols) binding with AChE, ABAD and BACE1 proteins. Docking experiment of fisetin with these proteins using two different tools namely iGEMDOCK and FlexX show significant binding with acceptable binding values. Thus, the potential inhibitory role of fisetin with AD associated proteins is documented.     

Refolding and functional assembly of the Vibrio cholerae porin OmpU recombinantly expressed in the cytoplasm of Escherichia coli

OmpU is one of the major outer membrane porins of Vibrio cholerae. OmpU has been biochemically characterized previously for its 'porin'-property. However, previous studies have used the OmpU protein extracted from the bacterial outer membrane envelope fractions. Such method of isolation imposes limitations on the availability of the protein reagent, and also enhances the possibility of the OmpU preparation being contaminated with lipid molecules of bacterial outer membrane origin, especially lipopolysaccharides (LPS). Here we report a strategy of purifying the V. cholerae OmpU protein recombinantly overexpressed in heterologous protein expression system in Escherichia coli, without its being incorporated into the bacterial membrane fraction. In our strategy, the majority of the protein was expressed as insoluble inclusion body in the E. coli cytoplasm, the protein was dissolved by denaturation in 8M urea, refolded, and purified to homogeneity in presence of detergent. Our strategy allowed isolation of the recombinant OmpU protein with significantly enhanced yield as compared to that of the wild type protein extracted from the V. cholerae membrane fraction. The recombinant V. cholerae OmpU protein generated in our study displayed functional channel-forming property in the synthetic liposome membrane, thus confirming its 'porin'-property. To the best of our knowledge, this is the first report showing an efficient refolding and functional assembly of the V. cholerae OmpU porin recombinantly expressed as inclusion body in the cytoplasm of a heterologous host E. coli.     

A scaled-down model for the translation of bacteriophage culture to manufacturing scale

Therapeutic bacteriophages are emerging as a potential alternative to antibiotics and synergistic treatment of antimicrobial-resistant infections. This is reflected by their use in an increasing number of recent clinical trials. Many more therapeutic bacteriophage is being investigated in preclinical research and due to the bespoke nature of these products with respect to their limited infection spectrum, translation to the clinic requires combined understanding of the biology underpinning the bioprocess and how this can be optimized and streamlined for efficient methods of scalable manufacture. Bacteriophage research is currently limited to laboratory scale studies ranging from 1–20 ml, emerging therapies include bacteriophage cocktails to increase the spectrum of infectivity and require multiple large-scale bioreactors (up to 50 L) containing different bacteriophage–bacterial host reactions. Scaling bioprocesses from the milliliter scale to multi-liter large-scale bioreactors is challenging in itself, but performing this for individual phage-host bioprocesses to facilitate reliable and robust manufacture of phage cocktails increases the complexity. This study used a full factorial design of experiments approach to explore key process input variables (temperature, time of infection, multiplicity of infection, agitation) for their influence on key process outputs (bacteriophage yield, infection kinetics) for two bacteriophage–bacterial host bioprocesses (T4 – Escherichia coli; Phage K – Staphylococcus aureus). The research aimed to determine common input variables that positively influence output yield and found that the temperature at the point of infection had the greatest influence on bacteriophage yield for both bioprocesses. The study also aimed to develop a scaled down shake-flask model to enable rapid optimization of bacteriophage batch bioprocessing and translate the bioprocess into a scale-up model with a 3 L working volume in stirred tank bioreactors. The optimization performed in the shake flask model achieved a 550-fold increase in bacteriophage yield and these improvements successfully translated to the large-scale cultures.     

Variations in vinblastine production at different stages of somatic embryogenesis,embryo, and field-grown plantlets of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> L. (G) Don,as revealed by HPLC

Catharanthus roseus L. (G) Don. is an important dicotyledonous medicinal plant that produces anticancer compounds, which are used for the treatment of a wide variety of cancers. We have quantified vinblastine (a major dimeric anticancer compound) in various in vitro raised tissues; embryogenic and nonembryogenic calli, three different embryogenic stages (proliferated, matured, and germinating embryo), somatic embryo derived plantlets and in ex vitro grown plantlets by using high performance liquid chromatography. Of the various obtained callus lines and embryogenesis stages, maximum vinblastine content was found in leaf callus and in germinating embryos. The leaves of somatic embryo-derived plantlets contained more vinblastine than did Catharanthus leaves developed ex vitro. The yield of vinblastine was monitored for 30 wk. The production of vinblastine appeared to be age dependent and tissue specific; the finding of our analyses is discussed in detail.     

Folic acid supplementation in pregnancy and implications in health and disease

Maternal exposure to dietary factors during pregnancy can influence embryonic development and may modulate the phenotype of offspring through epigenetic programming. Folate is critical for nucleotide synthesis, and preconceptional intake of dietary folic acid (FA) is credited with reduced incidences of neural tube defects in infants. While fortification of grains with FA resulted in a positive public-health outcome, concern has been raised for the need for further investigation of unintended consequences and potential health hazards arising from excessive FA intakes, especially following reports that FA may exert epigenetic effects. The objective of this article is to discuss the role of FA in human health and to review the benefits, concerns and epigenetic effects of maternal FA on the basis of recent findings that are important to design future studies.