A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

Searching to combine technologies for safer food attainment

Campylobacteriosis is an infection frequently acquired through the consumption of animal origin products. Chicken can be considered the main responsible cause in the transmission chain of this disease. Ionizing radiation was used to verify the reduction of the microbiological load of Campylobacter jejuni present in chicken liver, which, in natura, can present contamination in up to 100% of the cases. The doses of irradiation used were: 0.20 kGy, 0.27 kGy, 0.30 kGy and 0.35 kGy. The samples of chicken liver were acquired in aviaries, local supermarkets and large chain supermarkets. The samples were analyzed for Campylobacter at FIOCRUZ. Irradiation was performed at COPPE/UFRJ, using a Gamma Cell Irradiator with a 60Co gamma source. Only the frozen sample acquired at the local supermarket did not contain the bacterium. Campylobacter sp. was present in all other samples, even when using procedures and technologies that aimed at the impediment of the presence of this bacterium in food and, consequently, at the protection of human health. On the whole, the results were satisfactory; nevertheless, it is known that the bacterial growth conditions required by this bacterium are uncommon when compared to other enteropathogenic bacteria.

Postharvest technologies for mangosteen (Garcinia mangostana L.) conservation

The application of technologies to extend the postharvest life of mangosteen fruit was studied and compared to storage at 25 °C/70-75%R.H (25 °C control treatment). The fruits were packed in expanded polystyrene (EPS) trays (5 fruits/tray). Five treatments were carried out at 13 °C/ 90-95% RH: application of carnauba wax coating, lecithin + CMC (carboxymethyl cellulose) coating, 50 µm LDPE (low density polyethylene) film coating, 13 µm PVC (Polyvinyl chloride), and non-coated sample (13 °C control treatment). Physicochemical analyses were performed twice a week. A statistical design was completely randomized with 8 repetitions for each treatment plus the control treatment. The results were submitted to variance analysis, and the averages compared by the Tukey test at 5% probability. Among the quality parameters analyzed, more significant differences were observed for weight loss, texture, and peel moisture content. The results showed that the maximum storage period for mangosteen at 25 °C is two weeks; while storage at13 °C can guarantee the conservation of this fruit for 25 days. Therefore, the treatment at 13 °C/90-95% RH without the use of coatings and films was more effective and economical.