HBcAg (hepatitis B core antigen) is a nanoplex bioproduct that has a great potential in the development of therapeutic drugs and vaccines. In the present study, a continuous-flow bead milling for the disruption of Escherichia coli was optimized and a direct recovery protocol to isolate the recombinant HBcAg from the unclarified E. coli disruptate was developed. The optimal condition for continuous-flow bead milling for the release of HBcAg from E. coli was achieved at a feed flow rate of 15 litres/h, biomass concentration of 10% [ww/v (wet weight/vol.)] and impeller tip speed of 14 m/s. The sucrose-density-gradient analysis showed that the particulate form of the HBcAg released by this optimal condition is still preserved. In the direct purification of HBcAg from the unclarified disruptate, the AE-EBAC (anion-exchange expanded-bed adsorption chromatography) technique was employed. A 54% adsorption and 50.7% recovery of HBcAg were achieved in this direct recovery process. The purity of HBcAg recovered was 49.8%, which corresponds to a purification factor of 2.0. ELISA showed that the HBcAg recovered is functionally active.
With the long-term goal of developing an ultra-sensitive microcantilever-based biosensor for versatile biomarker detection, new controlled bioreceptor-analytes systems are being explored to overcome the disadvantages of conventional ones. Gold (Au) microwires have been used as a probe to overcome the tolerance problem that occurs in response to changes in environmental conditions. However, the cytotoxicity of Au microwires is still unclear. Here, we examined the cytotoxicity of Au microwires systems using both commercial and as-synthesised Au microwires. In vitro experiments show that commercial Au microwires with an average quoted length of 5.6 µm are highly toxic against Gram-negative Escherichia coli (E. coli) at 50 µg/mL. However, this toxicity is due to the presence of CTAB surfactant not by the microwires. Conversely, the as-synthesised Au microwires show non-cytotoxicity even at the maximum viable concentration (330 µg/mL). These findings may lead to the development of potentially life-saving cytotoxicity-free biosensors for an early diagnostic of potential diseases.
This paper describes the development of an integrated system using a dry film resistant (DFR) microfluidic channel consisting of pulsed field dielectrophoretic field-flow-fractionation (DEP-FFF) separation and optical detection. The prototype chip employs the pulse DEP-FFF concept to separate the cells (Escherichia coli and Saccharomyces cerevisiae) from a continuous flow, and the rate of release of the cells was measured. The separation experiments were conducted by changing the pulsing time over a pulsing time range of 2⁻24 s and a flow rate range of 1.2⁻9.6 μ L min - 1 . The frequency and voltage were set to a constant value of 1 M Hz and 14 V pk-pk, respectively. After cell sorting, the particles pass the optical fibre, and the incident light is scattered (or absorbed), thus, reducing the intensity of the transmitted light. The change in light level is measured by a spectrophotometer and recorded as an absorbance spectrum. The results revealed that, generally, the flow rate and pulsing time influenced the separation of E. coli and S. cerevisiae. It was found that E. coli had the highest rate of release, followed by S. cerevisiae. In this investigation, the developed integrated chip-in-a lab has enabled two microorganisms of different cell dielectric properties and particle size to be separated and subsequently detected using unique optical properties. Optimum separation between these two microorganisms could be obtained using a longer pulsing time of 12 s and a faster flow rate of 9.6 μ L min - 1 at a constant frequency, voltage, and a low conductivity.
Induction strategies for the periplasmic production of recombinant human IFN-alpha2b (interferon-alpha2b) by recombinant Escherichia coli Rosetta-gami 2(DE3) were optimized in shake-flask cultures using response surface methodology based on the central composite design. The factors included in the present study were induction point, which related to the attenuance of the cell culture, IPTG (isopropyl beta-D-thiogalactoside) concentration and induction temperature. Second-order polynomial models were used to correlate the abovementioned factors to soluble periplasmic IFN-alpha2b formation and percentage of soluble IFN-alpha2b translocated to the periplasmic space of E. coli. The models were found to be significant and subsequently validated. The proposed induction strategies consisted of induction at an attenuance of 4 (measured as D600), IPTG concentration of 0.05 mM and temperature of 25 degrees C. The optimized induction strategy reduced inclusion-body formation as evidenced by electron microscopy and yielded 323.8 ng/ml of IFN-alpha2b in the periplasmic space with translocation of 74% of the total soluble product. In comparison with the non-optimized condition, soluble periplasmic production and the percentage of soluble IFN-alpha2b translocated to the periplasmic space obtained in optimized induction strategies were increased by approx. 20-fold and 1.4-fold respectively.
Recombinant human epidermal growth factor (EGF) was successfully expressed as a fusion protein in Escherichia coli system. This system was used OmpA signal sequence to produce soluble protein into the periplasm of E. coli. Human EGF (hEGF) synthesized in bacterial cell was found to be similar in size with the original protein and molecular weight approximately at 6.8 kDa. Cell proliferation assay was conducted to characterize the biological activity of hEGF on human dermal fibroblasts. The synthesized hEGF was found to be functional as compared with authentic hEGF in stimulating cell proliferation and promoting growth of cell. In comparison of biological activity between synthesized and commercial hEGF on cell proliferation, the results showed there was no significant different. This finding indicates the synthesized hEGF in E. coli system is fully bioactive in vitro.