An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation.

In this study, several expression strategies were investigated in order to develop a generic, highly productive and efficient protocol to produce nanobodies modified with a clickable alkyne function at their C-terminus via the intein-mediated protein ligation (IPL) technique. Hereto, the nanobody targeting the vascular cell adhesion molecule 1 (NbVCAM1) was used as a workhorse. The highlights of the protocol can be ascribed to a cytoplasmic expression of the nanobody-intein-chitin-binding domain fusion protein in the Escherichia coli SHuffle(®) T7 cells with a C-terminal extension, i.e. LEY, EFLEY or His6 spacer peptide, in the commonly used Luria-Bertani medium. The combination of these factors led to a high yield (up to 22 mg/l of culture) and nearly complete alkynation efficiency of the C-terminally modified nanobody via IPL. This yield can even be improved to ∼45 mg/l in the EnPresso(®) growth system but this method is more expensive and time-consuming. The resulting alkynated nanobodies retained excellent binding capacity towards the recombinant human VCAM1. The presented protocol benefits from time- and cost-effectiveness, which allows a feasible production up-scaling of generic alkynated nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to new biosurface applications that demand for a homogeneously oriented nanobody coupling. Prospectively, the alkynated nanobodies can be covalently coupled to a multitude of azide-containing counterparts, e.g. contrast labeling agents, particles or surfaces for numerous innovative applications.

Differences in metal sequestration between zebra mussels from clean and polluted field locations.

Organisms are able to detoxify accumulated metals by, e.g. binding them to metallothionein (MT) and/or sequestering them in metal-rich granules (MRG). The different factors involved in determining the capacity or efficiency with which metals are detoxified are not yet known. In this work we studied how the sub-cellular distribution pattern of cadmium, copper and zinc in whole tissue of zebra mussels from clean and polluted surface waters is influenced by the total accumulated metal concentration and by its physiological condition. Additionally we measured the metallothionein concentration in the mussel tissue. Metal concentration increased gradually in the metal-sensitive and detoxified sub-cellular fractions with increasing whole tissue concentrations. However, metal concentrations in the sensitive fractions did not increase to the same extent as metal concentrations in whole tissues. In more polluted mussels the contribution of MRG and MT became more important. Nevertheless, metal detoxification was not sufficient to prevent metal binding to heat-sensitive low molecular weight proteins (HDP fraction). Finally we found an indication that metal detoxification was influenced by the condition of the zebra mussels. MT content could be explained for up to 83% by variations in Zn concentration and physiological condition of the mussels.

A kinetic model for the relative contribution of waterborne and dietary cadmium and zinc in the common carp (Cyprinus carpio).

In this work, the uptake and whole-body accumulation of Cd and Zn by the common carp from water, diet, and a combination of both was studied using the radioactive tracers 09Cd and 65Zn. A three-compartment pharmacokinetic model was constructed and revealed metal- and exposure route-dependent accumulation profiles. Under the tested exposure conditions in water (0.1 microM Cd and 1 microM Zn), only 0.1 and 0.07%, respectively, of the total Cd and Zn load that passed the branchial surfaces was accumulated by the carp. In contrast, the carp accumulated 20 and 29% of the total ingested Cd and Zn load (mean concentrations in food of newly accumulated metals were 2.24 +/- 0.29 and 19.91 +/- 2.89 nmol/g wet weight, respectively). However, the contribution of the waterborne metals to the whole-body metal accumulation was higher than the metal uptake from food. The constructed model was used to simulate the effect of variable assimilation efficiency (AE) values on the relative importance of dietary Cd and Zn exposure to the overall metal accumulation in common carp. This simulation was performed under a realistic exposure scenario and with Cd and Zn AE values ranging from 5 to 95%. Dissolved Zn levels were higher and Cd levels lower compared to the laboratory experiments. Levels in the midge larvae were much higher. The results show that under these conditions, even at the lowest Cd and Zn AEs, almost 40% of the total body Cd and Zn concentrations originated from dietary Cd and Zn exposure. Taking into account the Cd and Zn AE of the laboratory experiments, respectively, 31 and 55%, more than 85% of the total body Cd and Zn was taken up from the food.

New metallothionein mRNAs in Gobio gobio reveal at least three gene duplication events in cyprinid metallothionein evolution.

This paper reports the identification and analysis of the primary structure of three novel metallothionein cDNA sequences in the gudgeon, Gobio gobio (Cyprinidae). Two different 180 bp coding regions were identified, resulting in two MT isoforms differing in one amino acid. The primary structure of the amino acid sequence was compared to other cyprinid MT sequences. Furthermore, two differently sized cDNAs were discovered in one of the two transcripts. We present a phylogenetic comparison of our sequences to other, previously published cyprinid MT gene sequences. Our analysis reveals an unexpected complexity in cyprinid MT evolution, with at least three gene duplication events. Differences and homologies between the evolution of cyprinid MT genes are compared to other teleost families. Finally, possible implications for metallothionein classification are discussed.

Dynamic model for the accumulation of cadmium and zinc from water and sediment by the aquatic oligochaete, Tubifex tubifex.

In aquatic environments, organisms are exposed to and accumulate metals via waterborne and dietary routes including ingested sediment. A key element in understanding metal uptake and accumulation is information concerning the relative importance of the routes of uptake and the kinetics of the processes. In this work the bioaccumulation of the essential element zinc and the nonessential element cadmium were studied from the aqueous and sediment phase, in the cosmopolitan oligochaete Tubifex tubifex, using the radiotracers 109Cd and 65Zn. A compartmental kinetic model was constructed and parametrized by fitting the model to metal body concentrations. Using the pharmacokinetic modeling approach and taking into account the distribution of the metal between water and sediment, the different routes were quantitatively separated. Under the experimental conditions, the sediment phase accounted for 9.8% of the cadmium and 52% of the zinc uptake. These values are based on the uptake of the radiotracers spiked sediments and therefore likely represent maximal values since it was shown that under the specific conditions this was the most mobile metal fraction. This difference was largely explained by the large difference in assimilation efficiency between cadmium and zinc. Simulations of different conditions showed that both dissolved and sediment-associated metal can be important sources of metal exposure for the worms and that the relative importance strongly depends on the metal and exposure conditions including the lability of the metals in the sediment phase.