Metal-dependent glycosylation in recombinant metallothioneins.

We show for the first time glycosylation of recombinant metallothioneins (MTs) produced in E. coli. Interestingly, our results show that the glycosylation level of the recombinant MTs is inversely proportional to the degree of protein structuration, and reflects their different metal preferences.

Single chain variable fragment fused to maltose binding protein: a modular nanocarrier platform for the targeted delivery of antitumorals.

The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due to their smaller size, low immunogenicity, and low-cost production. Although covalent strategies for the preparation of such ScFv-based therapeutic conjugates are prevalent, this approach is not straightforward, as it requires prior chemical activation and/or modification of both the ScFv and the therapeutics for the application of robust chemistries. A non-covalent alternative based on ScFv fused to maltose-binding protein (MBP) acting as a binding adapter is proposed for active targeted delivery. MBP-ScFv proves to be a valuable modular platform to synergistically bind maltose-derivatized therapeutic cargos through the MBP, while preserving the targeting competences provided by the ScFv. The methodology has been tested by using a mutated maltose-binding protein (MBP I334W) with an enhanced affinity toward maltose and an ScFv coding sequence toward the human epidermal growth factor receptor 2 (HER2). Non-covalent binding complexes of the resulting MBP-ScFv fusion protein with diverse maltosylated therapeutic cargos (a near-infrared dye, a maltosylated supramolecular ß-cyclodextrin container for doxorubicin, and non-viral polyplex gene vector) were easily prepared and characterized. In vitro and in vivo assays using cell lines that express or not the HER2 epitope, and mice xenografts of HER2 expressing cells demonstrated the capability and versatility of MBP-ScFv for diagnosis, imaging, and drug and plasmid active targeted tumor delivery. Remarkably, the modularity of the MBP-ScFv platform allows the flexible interchange of both the cargos and the coding sequence for the ScFv, allowing ad hoc solutions in targeting delivery without any further optimization since the MBP acts as a pivotal element.

Evidence of non-functional redundancy between two pea h-type thioredoxins by specificity and stability studies.

The largest group of plant thioredoxins (TRXs) consists of the so-called h-type; their great number raises questions about their specific or redundant roles in plant cells. Pisum sativum thioredoxin h1 (PsTRXh1) and Pisum sativum thioredoxin h2 (PsTRXh2) are both h-type TRXs from pea (Pisum sativum) previously identified and biochemically characterized. While both are involved in redox regulation and show a high-sequence identity (60%), they display different behavior during in vitro and in vivo assays. In this work, we show that these two proteins display different specificity in the capturing of protein targets in vitro, by the use of a new stringent method. PsTRXh2 interacted with classical antioxidant proteins, whereas PsTRXh1 showed a completely different pattern of targeted proteins, and was able to capture a transcription factor. We also showed that the two proteins display very different thermal and chemical stabilities. We suggest that the differences in thermal and chemical stability point to a distinct and characteristic pattern of protein specificity.