Purification of long helical capsid of newcastle disease virus from Escherichia coli using anion exchange chromatography

NP_Δc375 is a truncated version of the nucleocapsid protein of Newcastle disease virus (NDV) which self‐assembles into a long helical structure. A packed bed anion exchange chromatography (PB‐AEC), SepFastTM Supor Q pre‐packed column, was used to purify NP_Δc375 from clarified feedstock. This PB‐AEC column adsorbed 76.2% of NP_Δc375 from the clarified feedstock. About 67.5% of the adsorbed NP_Δc375 was successfully eluted from the column by applying 50 mM Tris‐HCl elution buffer supplemented with 0.5 M NaCl at pH 7. Thus, a recovery yield of 51.4% with a purity of 76.7% which corresponds to a purification factor of 6.5 was achieved in this PB‐AEC operation. Electron microscopic analysis revealed that the helical structure of the NP_Δc375 purified by SepFast^TM Supor Q pre‐packed column was as long as 490 nm and 22–24 nm in diameter. The antigenicity of the purified NP_Δc375 was confirmed by enzyme‐linked immunosorbent assay. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 564–567, 2013     

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

The disruption ofSaccharomyces cerevisiae cells and release of glucose 6-phosphate dehydrogenase (G6PDH) in a horizontal dyno bead mill operated in continuous recycling mode

Baker’s yeast was disrupted in a 1.4-L stainless steel horizontal bead mill under a continuous recycle mode using 0.3 mm diameter zirconia beads as abrasive. A single pass in continuous mode bead mill operation liberates half of the maximally released protein. The maximum total protein release can only be achieved after passaging the cells 5 times through the disruption chamber. The degree of cell disruption was increased with the increase in feeding rate, but the total protein release was highest at the middle range of feeding rate (45 L/h). The total protein release was increased with an increase in biomass concentration from 10 to 50% (w/v). However, higher heat dissipation as a result of high viscosity of concentrated biomass led to the denaturation of labile protein such as glucose 6-phosphate dehydrogenase (G6PDH). As a result the highest specific activity of G6PDH was achieved at biomass concentration of 20% (ww/v). Generally, the degree of cell disruption and total protein released were increased with an increase in impeller tip speed, but the specific activity of G6PDH was decreased substantially at higher impeller tip speed (14 m/s). Both the degree of cell disruption and total protein release increased, as the bead loading increased from 75 to 85% (v/v). Hence, in order to obtain a higher yield of labile protein such as G6PDH, the yeast cell should not be disrupted at biomass concentration and impeller tip speed higher than 20% (w/v) and 10 m/s, respectively.