Mathematical modeling of elution curves for a protein mixture in ion exchange chromatography and for the optimal selection of operational conditions

Elution curves in ionic exchange chromatography (IEC) for a three-protein mixture (alpha-lactoalbumin, ovalbumin, and beta-lactoglobulin), carried out under different flow rates and ionic strength conditions, were simulated using two different mathematical models. These models were the Plate Model and the more fundamentally based Rate Model. Relatively low protein concentrations were used to avoid protein-protein interactions. Simulated elution curves were compared with experimental data not used for parameter identification. Deviation between experimental data and the simulated curves using the Plate Model was less than 0.0189 (absorbance units); a slightly higher deviation [0.0252 (absorbance units)] was obtained when the Rate Model was used. A cost function was built that included the effect of the different production stages, namely fermentation, purification, and concentration. These considered the effect on the performance of IEC; yield, purity, concentration and the time needed to accomplish the separation. Operational conditions in the IEC such as flow rate, ionic strength gradient and the operational time can be selected using this model in order to find the minimum cost for the protein production process depending on the characteristics of the final product desired such as purity and yield. This cost function was successfully used for the selection of the operational conditions as well as the fraction of the product to be collected (peak cutting) in IEC. It can be used for protein products with different characteristics and qualities, such as purity and yield, by choosing the appropriate parameters.     

Human Fidgetin is a microtubule severing the enzyme and minus-end depolymerase that regulates mitosis

Fidgetin is a member of the AAA protein superfamily with important roles in mammalian development. Here we show that human Fidgetin is a potent microtubule severing and depolymerizing the enzyme used to regulate mitotic spindle architecture, dynamics and anaphase A. In vitro, recombinant human Fidgetin severs taxol-stabilized microtubules along their length and promotes depolymerization, primarily from their minus-ends. In cells, human Fidgetin targets to centrosomes, and its depletion with siRNA significantly reduces the velocity of poleward tubulin flux and anaphase A chromatid-to-pole motion. In addition, the loss of Fidgetin induces a microtubule-dependent enlargement of mitotic centrosomes and an increase in the number and length of astral microtubules. Based on these data, we propose that human Fidgetin actively suppresses microtubule growth from and attachment to centrosomes.     

PEG activation and ligand binding for the affinity partitioning of proteins in aqueous two-phase systems

Summary PEG has been activated using epoxy-oxirane, epichlorohydrin and periodate based reactions. The coupling to activated PEG of several protein ligands of different sizes was investigated. Glutathione, trypsin inhibitor, Protein A and anti-BSA have been bound to PEG and used to increase the selectivity of protein separation in aqueous two-phase systems.     

The isolation of lytic enzymes from Cytophaga and their application to the rupture of yeast cells

Yeast–lytic enzymes have been isolated on a pilot scale from Cytophaga species by precipitation and the light, enzyme-rich solid phase recovered by liquid-liquid separation. The enzyme complex was immobilized to soluble polymeric carbohydrates and the effectiveness of the free and immobilized enzyme for protein release and cell debris dissolution has been assessed.     

Hunting for cultivable <Emphasis Type="Italic">Micromonospora</Emphasis> strains in soils of the Atacama Desert

Innovative procedures were used to selectively isolate small numbers of Micromonospora strains from extreme hyper-arid and high altitude Atacama Desert soils. Micromonosporae were recognised on isolation plates by their ability to produce filamentous microcolonies that were strongly attached to the agar. Most of the isolates formed characteristic orange colonies that lacked aerial hyphae and turned black on spore formation, whereas those from the high altitude soil were dry, blue-green and covered by white aerial hyphae. The isolates were assigned to seven multi- and eleven single-membered groups based on BOX-PCR profiles. Representatives of the groups were assigned to either multi-membered clades that also contained marker strains or formed distinct phyletic lines in the Micromonospora 16S rRNA gene tree; many of the isolates were considered to be putatively novel species of Micromonospora. Most of the isolates from the high altitude soils showed activity against wild type strains of Bacillus subtilis and Pseudomonas fluorescens while those from the rhizosphere of Parastrephia quadrangulares and from the Lomas Bayas hyper-arid soil showed resistance to UV radiation.     

Global gene expression in recombinant and non-recombinant yeast Saccharomyces cerevisiae in three different metabolic states

Global gene expression of two strains of Saccharomyces cerevisiae, one recombinant (P+), accumulating large amounts of an intracellular protein Superoxide Dismutase (SOD) and one non-recombinant (P−) which does not contain the recombinant plasmid, were compared in batch culture during diauxic growth when cells were growing exponentially on glucose, when they were growing exponentially on ethanol, and in the early stationary phase when glycerol was being utilized.When comparing the gene expression for P− (and P+) during growth on ethanol to that on glucose (Eth/Gluc), overexpression is related to an increase in consumption of glycerol, activation of the TCA cycle, degradation of glycogen and metabolism of ethanol. Furthermore, 97.6% of genes (80 genes) involved in the central metabolic pathway are overexpressed. This is similar to that observed by DeRisi et al. [DeRisi, J.L., Iyer, V.R. & Brown, P.O. 1997. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278:680–686.] but very different from was observed for Metabolic Flux Analysis (MFA), where the specific growth rate is lowered to ca. 40%, the fluxes in the TCA cycle are reduced to ca. 40% (to 30% in P+), glycolysis is reduced to virtually 0 and protein synthesis to ca. 50% (to 40% in P+). Clearly it is not possible to correlate in a simple or direct way, quantitative mRNA expression levels with cell function which is shown by the Metabolic Flux Analysis (MFA).When comparing the two strains in the 3 growth stages, 4 genes were found to be under or overexpressed in all cases. The products of all of these genes are expressed at the plasma membrane or cell wall of the yeast. While comparing the strains (P+/P−) when growing on glucose, ethanol and in the early stationary phase, many of the genes of the central metabolic pathways are underexpressed in P+, which is similar to the behaviour of the metabolic fluxes of both strains (MFA). Comparing the gene expression for P− (and to some extent P+) during the early stationary phase to growth on ethanol (Stat/Eth), underexpression is generalized. This shows that the switch in metabolism between ethanol and early stationary phases has an almost instantaneous effect on gene expression but a much more retarded effect on metabolic fluxes and that the “early stationary” phase represents a “late ethanol” phase from the metabolic analysis point of view since ethanol is still present and being consumed although at a much slower rate.     

Monoclonal antibody to dengue capsid protein: Its application in dengue studies

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.Key words: monoclonal antibodies, capsid protein, dengue virus, diagnosis, immunoassays     

Diversity of culturable actinomycetes in hyper-arid soils of the Atacama Desert,Chile

The Atacama Desert presents one of the most extreme environments on Earth and we report here the first extensive isolations of actinomycetes from soils at various locations within the Desert. The use of selective isolation procedures enabled actinomycetes to be recovered from arid, hyper-arid and even extreme hyper-arid environments in significant numbers and diversity. In some cases actinomycetes were the only culturable bacteria to be isolated under the conditions of this study. Phylogenetic analysis and some phenotypic characterisation revealed that the majority of isolates belonged to members of the genera Amycolatopsis, Lechevalieria and Streptomyces, a high proportion of which represent novel centres of taxonomic variation. The results of this study support the view that arid desert soils constitute a largely unexplored repository of novel bacteria, while the high incidence of non-ribosomal peptide synthase genes in our isolates recommend them as promising material in screening for new bioactive natural products.