Purification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatography

The current study explores the possibility of using a polyethyleneglycol(PEG)/ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400, 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20 % (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16 % w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35 % w/w PEG 600, 6% w/w NH4)2SO4), it was possible to recover 100 % of pDNA in the bottom phase with a 3-fold increase in concentration. Further increase in the lysate load up to 40 %(w/w) with this system resulted in a 8-fold increase in pDNA concentration, but with a yield loss of 15 %. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with isopropanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.Portuguese Ministry of Science and Technology - POCTI/BIO/47245/2002

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

On the dual effect of glucose during production of pBAD/AraC-based minicircles

Minicircles are promising vectors for DNA vaccination, gene or cell therapies due to their increased transfection efficacy and transgene expression. The in vivo production of these novel vectors involves the arabinose inducible excision of a parental molecule into a minicircircle and a miniplasmid bacterial backbone. Tight control of recombination is crucial to maximize minicircle yields and purity. In this work, a minicircle production system was constructed that relies on the enzymatic activity of ParA resolvase, a recombinase that is expressed under the transcription control of the arabinose inducible expression system pBAD/AraC, and on Escherichia coli BWAA, a strain improved for arabinose uptake. Undesired recombination already after 4 h of incubation in Luria-Bertani broth at 37 °C was observed due to the leaky expression from pBAD/AraC. While addition of glucose to the growth media repressed this leaky expression, it triggered a pH drop to 4.5 during exponential phase in shake flasks, which suppressed growth and plasmid production. The quantitative PCR analysis confirmed only few copies of high-copy number plasmid inside of the E. coli cells. To ensure the stability of minicircle-producing system, seed cultures should be grown at 30 °C with glucose overnight whereas cells for minicircle production should be grown in shake flasks at 37 °C without glucose up to early stationary phase when the recombination is induced by addition of arabinose.MIT-Portugal ProgramFundacao para a Ciencia e a Tecnologia (PhD Grant SFRH/BD/33786/2009)Fundacao para a Ciencia e a Tecnologia (Project PTDC/EBB-EBI/113650/2009

Engineering of Escherichia coli strains for plasmid biopharmaceutical production: Scale-up challenges

Plasmid-based vaccines and therapeutics have been making their way into the clinic in the last years. The existence of cost-effective manufacturing processes capable of delivering high amounts of high-quality plasmid DNA (pDNA) is essential to generate enough material for trials and support future commercialization. However, the development of pDNA manufacturing processes is often hampered by difficulties in predicting process scale performance of Escherichia coli cultivation on the basis of results obtained at lab scale. This paper reports on the differences observed in pDNA production when using shake flask and bench-scale bioreactor cultivation of E. coli strains MG1655ΔendAΔrecA and DH5α in complex media with 20 g/L of glucose. MG1655ΔendAΔrecA produced 5-fold more pDNA (9.8 mg/g DCW) in bioreactor than in shake flask (1.9 mg/g DCW) and DH5α produced 4-fold more pDNA (8 mg/g DCW) in bioreactor than in shake flask (2 mg/g DCW). Accumulation of acetate was also significant in shake flasks but not in bioreactors, a fact that was attributed to a lack of control of pH.MIT-Portugal ProgramFundacao para a Ciencia e a Tecnologia (Project PTDC/EBB-EBI/113650/2009)Fundacao para a Ciencia e a Tecnologia (PhD Grant SFRH/BD/33786/2009

Imaging of Genetically Encoded FRET-Based Biosensors to Detect GPCR Activity

A wealth of assays for screening GPCR activity have been developed. Biosensors that employ Förster Resonance Energy transfer (FRET) are specific and enable dynamic measurements. Moreover, FRET biosensors are ideally suited for the analysis of single living cells. The FRET biosensors described in this manuscript are entirely genetically encoded by plasmids. Here, protocols for employing FRET-based biosensors to detect G protein activity upon GPCR activation are reported. The protocols include details on the isolation of plasmids, transfection, generation of stable cell lines with the FRET biosensors, FRET ratio imaging, and data analysis

A single-particle plasmon sensor to monitor proteolytic activity in real-time

We have established a label-free plasmonic platform that monitors proteolytic activity in real-time. The sensor consists of a random array of gold nanorods that are functionalized with a design peptide that is specifically cleaved by thrombin resulting in a blue-shift of the longitudinal plasmon. By monitoring the plasmon of many individual nanorods we determined thrombin’s proteolytic activity in real-time and inferred relevant kinetic parameters. Furthermore, comparison to a kinetic model revealed that the plasmon shift is dictated by a competition between peptide cleavage and thrombin binding, which have opposing effects on the measured plasmon shift. The dynamic range of the sensor is greater than two orders of magnitude, and it is capable of detecting physiologically relevant levels of active thrombin down to 3 nM in buffered conditions. We expect these plasmon-mediated label-free sensors open the window to a range of applications stretching from diagnostic and characterization of bleeding disorders to fundamental proteolytic and pharmacological studies

Rational engineering of Escherichia coli strains for plasmid biopharmaceutical manufacturing

Plasmid DNA (pDNA) has become very attractive as a biopharmaceutical, especially for gene therapy and DNA vaccination. Currently, there are a few products licensed for veterinary applications and numerous plasmids in clinical trials for use in humans. Recent work in both academia and industry demonstrates a need for technological and economical improvement in pDNA manufacturing. Significant progress has been achieved in plasmid design and downstream processing, but there is still a demand for improved production strains. This review focuses on engineering of Escherichia coli strains for plasmid DNA production, understanding the differences between the traditional use of pDNA for recombinant protein production and its role as a biopharmaceutical. We will present recent developments in engineering of E. coli strains, highlight essential genes for improvement of pDNA yield and quality, and analyze the impact of various process strategies on gene expression in pDNA production strains. Keywords: Central metabolism; Escherichia coli; Metabolic engineering; Plasmid biopharmaceuticals; Strain engineerin

Enhanced-Fluorescence of a Dye on DNA- assembled Gold Nano-Dimers Discriminated by Lifetime Correlation Spectroscopy

We have employed DNA-directed assembly to prepare dimers of gold nanoparticles and used their longitudinally coupled plasmon mode to enhance the fluorescence emission of an organic red-emitting dye, Atto-655. The plasmon- enhanced fluorescence of this dye using dimers of 80 nm particles was measured at single molecule detection level. The top enhancement factors were above 1000-fold in 71% of the dimers within a total of 32 dimers measured, and, in some cases, they reached almost 4000-fold, in good agreement with model simulations. Additionally, fluorescence lifetime correlation analysis enabled the separation of enhanced from non-enhanced emission simultaneously collected in our confocal detection volume. This approach allowed us to recover a short relaxation component exclusive to enhanced emission that is attributed to the interaction of the dye with DNA in the interparticle gaps. </div