Efficient Disruption of Escherichia coli for Plasmid DNA Recovery in a Bead Mill

Adriana Padilla-Zamudio,J Lucero-Acuña,Patricia Guerrero-Germán,Armando Tejeda-Mansir,Jaime Ortega-López

doi:10.3390/app8010030

Adriana Padilla-Zamudio, J Lucero-Acuña + Show 3 more

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https://doi.org/10.3390/app8010030

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Abstract

The release kinetics of pDNA in a bead mill was studied. Samples taken during the process were analyzed to determine total pDNA (pDNA(t)) and supercoiled pDNA (pDNA(sc)) concentration. In order to identify important variables of the process and to develop an empirical model for optimal pDNA(t) and pDNA(sc) release, a two level 23 factorial design was used with variables: mill frequency, cell concentration, and bead size. The results were analyzed by response surface methodology. The optimized conditions for pDNA(t) yield 13.26 mg/g dcw (93.41% recovery), with a mill frequency of 30 Hz, a bead size of 0.10–0.25 mm, and a cell concentration of 20 g wcw/L. However, the optimized conditions for pDNA(sc) yield 7.65 mg/g dcw (92.05% recovery), with a mill frequency of 15 Hz, a bead size of 0.10–0.25 mm, and a cell concentration of 10 g wcw/L. Cell disruption in a bead mill was proved efficient for the release of pDNA(t) and pDNA(sc) compared to the alkaline treatment. The results obtained suggest a compromise between pDNA(sc) purity and recuperation in the process development.

Highlights

IntroductionInterest in the use of pDNA vaccines has strongly increased [1]
During the last decade, interest in the use of pDNA vaccines has strongly increased [1]
Line 8 shows the electrophoretic analysis of a lysate sample obtained by alkaline treatment

Summary

Introduction

Interest in the use of pDNA vaccines has strongly increased [1]. The pDNA vaccines are believed to be potentially useful in a wide range of applications including preventive and therapeutic vaccines for viral, bacterial, and parasitic diseases as well as cancer [2,3]. The production of pDNA follow the steps of fermentation, primary and intermediate recovery, and purification. A sequence of operations is followed to recover pDNA molecules from the host cells and to remove impurities and contaminants from the lysate, until the desired level of purity and other specifications are obtained [4]. Cell disruption is the first and the most critical step in the bioprocess since this step influences the yield and quality of the product [5,6].

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