Molecuiar evolution of cytochrome c from invertebrates

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Production of adenoviral vectors in 293 cells: a case study of the adaptation of attached cells to grow in suspension

A study of the production of adenoviral vectors in suspension 293 cells has been explored. A defined serumfree medium (293 SFM II) formulated without human or animal origin components from Invitrogen was used for the suspension adapted 293 cells. It was demonstrated that the 293 cells can be adapted to grow in suspension using serum free medium. The effect of different cell culture parameters was determined. The production technique demonstrated here is expected to simplify purification processes and circumvents the problems associated with serum containing medium

Physical characterisations of a single-stage Kühni-type aqueous two-phase extraction column

The main parameters which influence the behaviour of phase separation in a single-stage Kühni-type aqueous two-phase extraction column containing polyethylene (PEG) and di-potassium hydrogen phosphate were characterised. Two aqueous two-phase system (ATPS) composed of 12% (w/w) PEG 1450 and 12% (w/w) di-potassium hydrogen phosphate (designated as 12/12) and 12% (w/w) PEG 1450 and 11% (w/w) di-potassium hydrogen phosphate (designated as 12/11) were chosen in this study. The hold-up D increased with increasing impeller speeds and mobile phase flow rates. Phase separation for the 12/11 system was slower than that for the 12/12 system, which resulted in higher dispersed phase hold-up values for the 12/11 system. For 12/12 system, mass transfer of plasmid DNA (pDNA) from the dispersed mobile phase to the stationary phase increased rapidly with increasing impeller speeds of 130, 160 and 200 rpm which was reflected in the decreased values for CT/CTo. The degree of back-mixing quantified by the axial dispersion coefficient Dax was estimated to be 2.7 × 10−6 m2 s−1

The comparative recovery performance of anion exchange and dye-ligand fluidised bed adsorption of G3PDH from unclarified yeast extract

The comparative recovery performance of anion exchange and dye ligand fluidised bed adsorption of intracellular enzyme, glyceraldehydes 3-phosphate dehydrogenase (G3PDH) from unclarified disrupted yeast has been undertaken. The commercially available anion exchanger, Streamline QXL and the kiesleguhr-agarose composite adsorbent, Microsorb K6AX derived with dye-ligand (Cibaron Blue 3GA) were employed in fluidized bed experiments. The adsorbents were evaluated in respect of recovery performance in terms of yield, purity and enzyme specific activity

Selective partition of plasmid DNA and RNA in aqueous two-phase systems by the addition of neutral salt.

The selective partition of plasmid DNA (pDNA) and RNA in polyethylene glycol (PEG) and di-potassium hydrogen phosphate aqueous two-phase systems (ATPSs) by addition of NaCl salt was studied with pure pDNA and RNA solutions. The pDNA is increasingly excluded from top phases upon the addition of 0.5% and 3% (w/w) NaCl. With 3% (w/w) NaCl, the logarithmic partition coefficient of RNA was 1.2 and as a result, the RNA concentration in the top phase was 3.3-fold higher than that in the bottom phase. It is demonstrated that 47%, 13.7% and 7.5% (w/v) of PEG were required to achieve identical precipitation effects with PEG 300, 1450 and 6000, respectively. The precipitation efficiency of 6.3% (w/v) PEG 300 corresponds to that of 1% (w/v) PEG 6000. The excluded volume effects in the top phase were probably responsible for the selective exclusion of different nucleic acids species. The results obtained in this study contribute to the basic knowledge of partition of macromolecules in ATPSs in terms of excluded volume theory

Partition of plasmid DNA in polymer-salt aqueous two phase systems

The partition of plasmid DNA (pDNA) in polyethylene glycol (PEG)–phosphate aqueous two-phase systems (ATPS) is presented. A high molecular weight (HMW) and a low molecular weight (LMW) polymer, PEG-1450 and -300, were used in combination with di-potassium hydrogen phosphate. The experimental results demonstrated that the plasmid pTX0161 displays a varied partition behaviour in PEG–phosphate ATPS. In HMW PEG (PEG-1450–phosphate systems), pDNA partitioned to the bottom phase only. In LMW PEG (PEG-300–phosphate systems), pDNA partitioned to all of the phases with respect to the phase composition, system temperature and concentration of lysate used in the ATPS. In systems with volume ratios higher than one, pDNA was mainly recovered in the top phase. For volume ratios between 0.5 and 1, pDNA mainly partitioned to the interface. In systems with volume ratios below 0.5, most of the pDNA was recovered in the bottom phase. For temperatures between 4 and 25°C, the partition to the top phase decreased whereas partition to the interface steadily increased. At 25°C, over 80% of pDNA was recovered in the interface. The partition to the bottom phase increased steadily with increasing temperatures up to 40°C and the partition to the interface decreased. At 20°C, the recovery of pDNA in the interface gradually increased and reached a maximum at 60% (w/w) lysate with 80% recovery recorded. At 25°C, over 80% of pDNA was recovered in the interface from lysate concentrations greater than 35% (w/w). At 30°C, the top phase preference changed to an interface preference between 0 and 20% (w/w) lysate

Integration of mechanical cell disruption and fluidised bed recovery of G3PDH from unclarified disrupted yeast: a comparative study of the performance of unshielded and polymer shielded dye-ligand chromatography systems

The development of a simplified process for the simultaneous disruption and direct selective purification of intracellular proteins from unclarified yeast disruptate has been investigated. The recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from baker's yeast was selected as a potential demonstration of the generic applicability and practical feasibility of this integrated technique. The application of an adsorbent characterised by high density (UpFront steel-agarose; ρ = 2.65 g ml−1) facilitated the combining of cell disruption operation (bead milling of 50% ww/v of yeast suspension at 7.2 l h−1) with fluidised bed dye-ligand (Cibacron Blue 3GA) adsorption operated immediately downstream of the disrupter. The adoption of a polymer shielded, dye-ligand technique advanced recovery efficiency. It was demonstrated that G3PDH could be recovered with a yield of 67.5% bound activity and a specific activity of 40.2 IU mg−1, after a single step elution with 0.15 M NaCl. The generic application of this approach has been evaluated

Effect of Pluronic F-68, 5% CO2 atmosphere, HEPES, and antibiotic-antimycotic on suspension adapted 293 cells

The influence of different parameters upon cell culture of serum-free adapted 293 cells including the surfactant Pluronic F-68, carbon dioxide (5% CO2 atmosphere), buffer HEPES and antibiotic-antimycotic has been explored. A defined serum-free medium (SFM) formulated without human or animal origin components from Invitrogen was used to grow the suspension adapted 293 cells. For all cell culture parameters cell density and viability of the suspension adapted 293 cells were monitored. The results indicated that the PF68 concentrations ranging from 0.05% to 0.2% can be used in the culture of the suspension adapted 293 cells since no negative effect upon either cell density or viability was detected. This will minimize the formation of aggregates during cell culture. It was demonstrated that neither the cell density nor the viability of the suspension adapted 293 cells were affected by the 5% CO2 atmosphere at the inoculation cell densities evaluated. The use of the buffer HEPES in the cultivation of suspension adapted 293 cells did not cause negative effects upon cell density and viability. The addition of HEPES makes more robust the culture to pH fluctuations. The antibiotic-antimycotic can be used when needed at concentrations of up to 50 IU/ml for the culture of this particular cell line, with no apparent effect upon cell growth. The results obtained will contribute to a basic understanding of the 293 cell culture in the 293 SFM II and to the process development of their culture in bioreactors for the expression of different products of biotechnology interest

Effect of polymer shielding on elution of G3PDH bound to dye-ligand adsorbent

Batch binding experiments were performed to assess the recovery performance of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) bound to the unshielded and polymer (polyvinyl pyrrolidone, PVP)-shielded dye-ligand (Cibacron Blue 3GA) adsorbent. The adoption of a polymer-shielded, dye-ligand technique facilitated the elution efficiency of bound G3PDH. It was demonstrated that the recovery of G3PDH using polymer-shielded dye-ligand adsorption yielded higher elution efficiency, at 60.5% and a specific activity of 42.3 IU/mg, after a low ionic strength elution (0.15 M NaCl). The unshielded dye-ligand yielded lower elution efficiency, at 6.5% and a specific activity of 10.2 IU/mg