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.     

Decoding the drivers of deep-time wetland biodiversity: insights from an early Permian tropical lake ecosystem

Wetlands are important to continental evolution, providing both arenas and refugia for emerging and declining biotas. This significance and the high preservation potential make the resulting fossiliferous deposits essential for our understanding of past and future biodiversity. We reconstruct the trophic structure and age of the early Permian Manebach Lake ecosystem, Germany, a thriving wetland at a time when the tropical biosphere faced profound upheaval in the peaking Late Palaeozoic Icehouse. Nine excavations, high-resolution spatiotemporal documentation of fossils and strata, and U–Pb radioisotopic dating of tuffs allow us to distinguish autogenic and allogenic factors shaping the limnic biocoenosis. The Manebach Lake was an exorheic, oxygen-stratified, perennial water body on the 10¹–10² km² scale, integrated into the catchment draining much of the European Variscides. Lake formation paralleled an Asselian regional wet climatic interval and benefited from rising base level due to post-Variscan half-graben tectonics. Stromatolite-forming cyanobacteria, bivalves, several crustaceans, amblypterids and xenacanthid sharks formed a differentiated biocoenosis in the lake. Fossil stomach remains and teeth prove the rare presence of acanthodians, branchiosaurs and large amphibians. The results indicate woody-debris-bearing lake littorals devoid of semi-aquatic and aquatic plants as places suitable for stromatolites to grow, underpin the model of declining freshwater-shark diversity in most Permian Variscan basins, demonstrate fish/amphibian ratios in limnic assemblages to measure lake perenniality and reveal taphonomic biases in lake taphocoenoses. Our outcomes call for more knowledge about the diversity, ecology and fossilization pathways of past limnic biotas, particularly microorganisms and actinopterygian fishes, to reconstruct deep-time continental ecosystems.     

Characterization of soy protein hydrolysates and influence of its iron content on monoclonal antibody production by a murine hybridoma cell line

A challenging aspect with the use of protein hydrolysates in commercial manufacturing processes of recombinant therapeutic proteins is their impacts on the protein production due to a lack of understanding of batch-to-batch variability. Soy hydrolysates variability and its impact on fed-batch production of a recombinant monoclonal antibody (mAb) expressed in Sp2/0 cells were studied using 37 batches from the same vendor. The batch-to-batch variability of soy hydrolysates impacted cell growth, titer and product quality. Physicochemical characterization of batches confirmed that soy hydrolysates are mainly a source of amino acids and peptides containing lower amounts of other components such as carbohydrates and chemical elements in cell culture media. Soy hydrolysates composition of different batches was consistent except for trace elements. Statistical analyses identified iron as a potential marker of a poor process performance. To verify this correlation, two forms of iron, ferric ammonium citrate and ferrous sulfate, were added to a batch of soy hydrolysates associated to a low level of iron during cell culture. Both forms of iron reduced significantly cell growth, mAb titer and increased level of the acidic charge variants of the mAb. Consequently, trace element composition of soy hydrolysates or of all incoming raw materials might lead to significant impacts on process performance and product quality and therefore need to be tightly controlled.