High yield expression of catalytically active USP18 using a Trigger Factor fusion system

ABSTRACT: BACKGROUND: Covalent linkage of the ubiquitin-like protein ISG15 interferes with viral infection and USP18 is the major protease which specifically removes ISG15 from target proteins. Thus, boosting ISG15 modification by protease inhibition of USP18 might represent a new strategy to interfere with viral replication. However, so far no heterologous expression system was available to yield sufficient amounts of catalytically active protein for high-throughput based inhibitor screens. RESULTS: High-level heterologous expression of USP18 was achieved applying a novel chaperone-based fusion system in E. coli. Pure protein was obtained in a single-step on IMAC via a His6-tag. The USP18 fusion protein exhibited enzymatic activity towards cell derived ISG15 conjugated substrates and efficiently hydrolyzed ISG15-AMC. Specificity towards ISG15 was shown by covalent adduct formation with ISG15 vinyl sulfone but not with ubiquitin vinyl sulfone. CONCLUSION: The results presented here show that a chaperone fusion system can provide high yields of proteins difficult to express. The USP18 protein obtained here is suited to setup high-throughput small molecule inhibitor screens and forms the basis for detailed biochemical and structural characterization.     

Overexpression of Several Arabidopsis Histone Genes Increases Agrobacterium-Mediated Transformation and Transgene Expression in Plants

The Arabidopsis thaliana histone H2A-1 is important for Agrobacterium tumefaciens–mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, results in decreased T-DNA integration into the genome of Arabidopsis roots, whereas overexpression of HTA1 increases transformation frequency. To understand the mechanism by which HTA1 enhances transformation, we investigated the effects of overexpression of numerous Arabidopsis histones on transformation and transgene expression. Transgenic Arabidopsis containing cDNAs encoding histone H2A (HTA), histone H4 (HFO), and histone H3-11 (HTR11) displayed increased transformation susceptibility, whereas histone H2B (HTB) and most histone H3 (HTR) cDNAs did not increase transformation. A parallel increase in transient gene expression was observed when histone HTA, HFO, or HTR11 overexpression constructs were cotransfected with double- or single-stranded forms of a gusA gene into tobacco (Nicotiana tabacum) protoplasts. However, these cDNAs did not increase expression of a previously integrated transgene. We identified the N-terminal 39 amino acids of H2A-1 as sufficient to increase transient transgene expression in plants. After transfection, transgene DNA accumulates more rapidly in the presence of HTA1 than with a control construction. Our results suggest that certain histones enhance transgene expression, protect incoming transgene DNA during the initial stages of transformation, and subsequently increase the efficiency of Agrobacterium-mediated transformation.     

Protein supply and nutritional status in nineteenth century Bavaria, Prussia and France

What determined regional height differences in the 19th century? We compare anthropometric evidence with production estimates of different food products and other economic variables. To this end, we concentrate on 179 rural regions and 29 towns in Bavaria (Southeast Germany). This regionally disaggregated level of analysis enables us to study the influence of the local supply of different food products on the nutritional status of the population, among which milk turned out particularly important. This result is tested and confirmed with regional data from Prussia and France.     

Ca2+ control of electrolyte permeability in plasma membrane vesicles from cat pancreas

Summary The influence of Ca²⁺ and other cations on electrolyte permeability has been studied in isolated membrane vesicles from cat pancreas.Ca²⁺ in the micromolar to millimolar concentration range, as well as Mg²⁺, Sr²⁺, Mn²⁺ and La³⁺ at a tested concentration of 10^–4 m, increased Na⁺ permeability when applied at the vesicle inside. When added to the vesicle outside, however, they decreased Na⁺ permeability. Ba²⁺ was effective from the outside but not from the vesicle inside.When Ca²⁺ was present at both sides of the membrane, Na⁺ efflux was not affected as compared to that in the absence of Ca²⁺. Monovalent cations such as Rb⁺, Cs⁺, K⁺, Tris⁺ and choline⁺ decreased Na⁺ permeability when present at the vesicle outside at a concentration range of 10 to 100mm. Increasing Na⁺ concentrations from 10 to 100mm at the vesicle inside increased Na⁺ permeability.The temperature dependence of Na⁺ efflux revealed that the activation energy increased in the lower temperature range (0 to 10°C) when Ca²⁺ was present at the outside or at both sides, but not when present at the vesicle inside only or in the absence of Ca²⁺.The results suggest that the Ca²⁺ outside effect is due to binding of calcium to negatively charged phospholipids with a consequent reduction of both fluidity and Na⁺ permeability of the membrane. The Ca²⁺-inside effect most likely involves interaction with proteins with consequent increase in Na⁺ permeability.The data are consistent with current hypotheses on secretagogue-induced fluid secretion in acinar cells of the pancreas according to which secretagogues elicit NaCl and fluid secretion by liberating Ca²⁺ from cellular membranes and by stimulating Ca²⁺ influx into the cell. The increased intracellular Ca²⁺ concentration in turn increases the contraluminal Na⁺ permeability which leads to NaCl influx. The luminal sodium pump finally transports Na⁺ ions into the lumen.     

Repressor synthesis in regulatory mutants of bacteriophage P22

Summary SDS-polyacrylamide gel analysis of P22-infected Salmonella has allowed identification of the c₂ repressor (MW 31,000) and study of repressor synthesis in regulatory mutants of P22. Repressor is synthesized in reduced amounts or is absent in infections with P22, c1#7, P22, c2 am08, P22 c3 am03, and P22 c3 am012, but is synthesized in markedly increased amounts in the virulent mutant, P22 virB3, and its component mutants, vx and k5. Higher levels of repressor are also found in the P22 cly 17 mutant.     

Untersuchungen zum Harnsäuremetabolismus vonLittorina littorea (Gastropoda)

Periwinkles, as typical inhabitants of sea-shores, are subjected to extreme changes of environmental conditions, which affect their excretion. InLittorina littorea uric acid, urea and ammonium were detected particularly in the kidney, but the only metabolite excreted was ammonium. Only the concentration of uric acid was dependent on the availability of water; decreasing periods of submersion during low tide and raised salinities caused a higher concentration of uric acid, while increasing periods of submersion and lowered salinities effected the opposite. Transfer of periwinkles within their intertidal habitat and laboratory experiments to test the effect of salinity showed that the concentration of uric acid in the kidney is adaptable. The dependence of uric acid concentration in the kidney on environmental conditions and the ammoniotelic excretion ofL. littorea are discussed with regard to its particular living conditions. It is suggested that uric acid serves as nitrogen depot and has a particular function in osmoregulation.