Variability and expression profile of the DRF1 gene in four cultivars of durum wheat and one triticale under moderate water stress conditions.

The dehydration responsive element binding (DREB) proteins are important transcription factors that contribute to stress endurance in plants triggering the expression of a set of abiotic stress-related genes. A DREB2-related gene, previously referred to as dehydration responsive factor 1 (DRF1) was originally isolated and characterized in durum wheat. The aim of this study was to monitor the expression profiles of three alternatively spliced TdDRF1 transcripts during dehydration experiments and to evaluate the effects of genetic diversity on the molecular response, using experimental conditions reflecting as closely as possible water stress perceived by cereals in open field. To investigate the effect of moderate water stress conditions, time-course dehydration experiments were carried out under controlled conditions in the greenhouse on four durum wheat and one triticale genotypes. Differences were observed in molecular patterns, thus, suggesting a genotype dependency of the DRF1 gene expression in response to the stress induced. The biodiversity of the transcripts of the DRF1 gene was explored in order to assess the level of polymorphism and its possible effects on structure and function of putative proteins. A total of nine haplotypes were identified in the sequences cloned, seven of which encompassing polymorphisms in exon 4, including the region codifying for the DNA binding Apetala2 (AP2) domain. The 3D structural models of the AP2 domain were generated by homology modelling using the variability observed. The polymorphisms analysed did not significantly affect the structural arrangement of the DNA binding domains, thus resulting compatible with the putative functionality.

Bacterial cytoplasm production of an EGFP-labeled single-chain Fv antibody specific for the HER2 human receptor.

The human epidermal growth factor receptor 2 (HER2) is the main diagnostic marker of breast and ovary cancers. Here, to obtain a rapid and sensitive immunodiagnostic tool a single-chain antibody (scFv800E6) specific for the HER2 was fused to the N-terminus of the enhanced green fluorescent protein (EGFP) by a flexible linker. The soluble production of the novel scFv800E6-EGFP protein in the cytoplasm of Escherichia coli was investigated at different induction temperatures (25, 30 and 37°C); the intrinsic fluorescent properties and the binding activity to HER2 positive tumour cells of the fusion protein were analysed. Western blotting and fluorescence analysis of SDS-PAGE revealed the presence of two scFv800E6-EGFP forms, with different mobility and optical properties, their ratio depending on the induction temperature. The fluorescent form maintained the optical fluorescence properties of EGFP and exhibited a binding activity to the HER2-expressing cells comparable to that of the non-fused scFv800E6. In addition, to provide an insight into the effect of the induction temperature on the molecular structure, the folding of the fusion protein was assessed at atomic level by performing molecular dynamics simulations of the homology-derived model of scFv800E6-EGFP at 300 K and 310 K. The comparison of the data collected at these two temperatures revealed that the higher temperature affects specific structural elements. To improve the production of the soluble and functional scFv800E6-EGFP protein, "in silico" results could be utilised for ad hoc design of the molecular structure.

Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems.

BACKGROUND: Aberrant signaling by ErbB-2 (HER 2, Neu), a member of the human Epidermal Growth Factor (EGF) receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen. METHODS: Herein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs) to ErbB-2 that involves their functional expression in (a) bacteria, (b) transient as well as stable transgenic tobacco plants, and (c) a newly developed cell-free transcription-translation system. RESULTS: An ScFv (ScFv800E6) was selected by cloning immunoglobulin sequences from murine hybridomas, and was expressed and fully functional in all the expression platforms, thereby representing the first ScFv to ErbB-2 produced in hosts other than bacteria and yeast. ScFv800E6 was optimized with respect to redox synthesis conditions. Different tags were introduced flanking the ScFv800E6 backbone, with and without spacer arms, including a novel Strep II tag that outperforms conventional streptavidin-based detection systems. ScFv800E6 was resistant to standard chemical radiolabeling procedures (i.e. Chloramine T), displayed a binding ability extremely similar to that of the parental monovalent Fab' fragment, as well as a flow cytometry performance and an equilibrium binding affinity (Ka approximately 2 x 10(8) M(-1)) only slightly lower than those of the parental bivalent antibody, suggesting that its binding site is conserved as compared to that of the parental antibody molecule. ScFv800E6 was found to be compatible with routine reagents for immunohistochemical staining. CONCLUSION: ScFv800E6 is a useful reagent for in vitro biochemical and immunodiagnostic applications in oncology, and a candidate for future in vivo studies.

Functional expression of a single-chain antibody specific for the HER2 human oncogene in a bacterial reducing environment.

Recombinant antibody fragments represent useful tools for cancer diagnosis and therapy. Aberrant expression of the HER2 receptor is implicated in metastatic breast and ovary cancers, two malignancies with a high prevalence in young women. In this study, we focussed on a single-chain fragment of variable antibody regions specific for HER2 (scFv800E6) that can be expressed in a functional form in the cytoplasm of Escherichia coli. ScFv800E6 was extracted from bacterial cultures following induction at different temperatures and purified. The yield of both soluble and insoluble forms was measured. We found that scFv800E6 was functional when expressed in the soluble fraction in the bacteria cytosol. In addition, scFv800E6 extracted from inclusion bodies was easily refolded and largely recovered its functionality. Thus, scFv800E6 is intrinsically capable of efficient and functional folding in a reducing environment and represents one of the few described antibody fragments with a framework well adapted for cytoplasmic expression.