Analysis and use of endogenous nuclease activities in Escherichia coli lysates during the primary isolation of plasmids for gene therapy

Monteiro, G. A.; Ferreira, Guilherme N. M.; Cabral, Joaquim M. S.; Prazeres, Duarte Miguel F.

doi:10.1002/(sici)1097-0290(1999)66:3<189::aid-bit7>3.3.co;2-q

Cited by 3 publications

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“…Continuously decreasing fluorescence of RNA bands with respect to resuspension time can be shown in an agarose gel, which could be better studied in order to set up a combination strategy with added recombinant RNase. The effect of endogenous RNase activities at the stage of cleared lysate (through enzymes that survived the alkaline lysis process) was harnessed to increase the resulting plasmid purity [ 18 ]. However, a collateral damage to the plasmid DNA was also clearly observed and therefore this strategy would have to be optimized to ensure reproducibility.…”

Section: Discussionmentioning

confidence: 99%

Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification

Shankar¹,

Schäffer²,

Schmeer³

et al. 2021

Microb Cell Fact

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Background The use of bovine-origin ribonucleases has been part of the standard protocol for plasmid DNA purification. As the field of gene therapy now enters the clinical stage, such enzymes need to be phased out or alternative purification protocols need to be developed to ensure product safety and regulatory compliance. The recombinant expression of bacterial RNase is fraught with toxicity problems making it a challenging enzyme to express. The current study describes a plasmid construct that allowed expression of barnase in Escherichia coli under co-expression of its native inhibitor barstar. Results The pure enzyme without the inhibitor barstar was exported to the extracellular space through the periplasm and then purified from the cell-free supernatant. Cation exchange chromatography was employed as a primary purification step. This was followed by hydrophobic interaction chromatography which resulted in a concentrated fraction of active enzyme. Although current levels of volumetric activity achieved are quite meagre (4 Kunitz units mL− 1), in principle its application to plasmid DNA purification could be proved. Currently, this is capable of processing small amounts (13 g) of bacterial biomass for plasmid production. Conclusions The current work focusses on the downstream purification strategies for a recombinant RNase and sets a framework for higher scale production if specific productivity is increased by optimal hosts and/or re-engineered plasmids. Also important is to curtail the massive enzyme loss during purification by cation exchange chromatography. Application of even a relatively small amount of recombinant RNase would contribute to greatly reducing the initial RNA levels in alkaline lysates thereby augmenting further downstream plasmid purification steps.

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Section: Discussionmentioning

confidence: 99%

Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification

Shankar¹,

Schäffer²,

Schmeer³

et al. 2021

Microb Cell Fact

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“…This decrease was accompanied by an increase in the oc isoform, indicating that sc isoform degradation starts with a single break of the phospho diester bond in one of the strands. A significant reduction in RNA content was again observed, particularly at room temperature and 4°C, most likely as a result of the action of E. coli RNases that remain active despite the harsh lysis conditions [24].…”

Section: Resultsmentioning

confidence: 99%

On the stability of plasmid DNA vectors during cell culture and purification

et al. 2007

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Gene therapy and DNA vaccination applications have increased the demand for highly purified plasmid DNA (pDNA) in the last years. One of the main problems related to the scale-up of pDNA purification is the degradation of the supercoiled (sc) isoforms during cell culture and multi-stage purification. In this work, a systematic study of the stability of two model plasmids (3,697 and 6,050 bp) during a mid-scale production process, which includes fermentation, alkaline lysis, isopropanol and ammonium sulphate precipitation and hydrophobic interaction chromatography, was performed. Results indicate that by extending cell culture (up to 26 h) and cell lysis (up to 2 h) it is possible to significantly reduce the amounts of RNA, without significantly compromising the yields of the sc pDNA isoform, a feature that could be conveniently exploited for downstream processing purposes. The stability of pDNA upon storage of E. coli pellets at different temperatures indicates that, differently from RNA, pDNA is remarkably stable when stored in cell pellets (>3 weeks at 4 degrees C, >12 weeks at -20 degrees C) prior to processing. With alkaline lysates, however, storage at -20 degrees C is mandatory to avoid sc pDNA degradation within the first 8 weeks. Furthermore, the subsequent purification steps could be carried out at room temperature without significant pDNA degradation. Since the unit operations and process conditions studied in this work are similar to those generally used for plasmid DNA production, the results presented here may contribute to improve the current knowledge on plasmid stability and process optimization.

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“…A desirable reduction in the amount of RNA in heat treated extracts was consistently observed by agarose gel electrophoresis and SYBR Green II staining. This could be caused by flocculation, chemical degradation of the RNA, or endogenous ribonuclease activity in the extract (Monteiro et al, 1999).…”

Section: Comparison Of Acidic Autolytic Extraction/thermal Flocculatimentioning

confidence: 99%

Plasmid DNA production combining antibiotic‐free selection, inducible high yield fermentation, and novel autolytic purification

Carnes

Hodgson

Luke

et al. 2009

Biotech & Bioengineering

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DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, we have developed autolytic Escherichia coli host strains that express endolysin (phage lambdaR) in the cytoplasm. Expression of the endolysin is induced during fermentation by a heat inducible promoter. The endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall; hence the cells remain alive and intact and can be harvested by the usual methods. The plasmid DNA is then recovered by autolytic extraction under slightly acidic, low salt buffer conditions and treatment with a low concentration of non-ionic detergent. Under these conditions the E. coli genomic DNA remains associated with the insoluble cell debris and is removed by a solid-liquid separation. Here, we report fermentation, lysis methods, and plasmid purification using autolytic hosts.

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Analysis and use of endogenous nuclease activities in Escherichia coli lysates during the primary isolation of plasmids for gene therapy

Cited by 3 publications

References 0 publications

Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification

Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification

On the stability of plasmid DNA vectors during cell culture and purification

Plasmid DNA production combining antibiotic‐free selection, inducible high yield fermentation, and novel autolytic purification

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