Plasmid Biopharmaceuticals

Prazeres, Duarte Miguel F.; Monteiro, Gabriel A.

doi:10.1128/9781555818982.ch34

Cited by 11 publications

(10 citation statements)

References 189 publications

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“…A semi‐defined medium (DM), consisting of salts, glucose, and casein hydrolysate, was shown to support a higher cell density and plasmid stability than the complex medium, LB, and plasmid extracted from cells grown on semi‐DM was of a higher quality with respect to supercoiling and gDNA level. There are other reports of fermentation strategies for the production of pDNA; A summary of those reported is given in Reference . Wang et al showed that plasmid stability was higher in a DM in comparison to two complex media.…”

Section: Introductionmentioning

confidence: 99%

The role of amino acids in the amplification and quality of DNA vectors for industrial applications

Dorward

O’Kennedy

Folarin

et al. 2019

Biotechnology Progress

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In this study, we have demonstrated that the type and feeding regimen of amino acids have a significant impact on the quality as well as the quantity of DNA vectors produced. Nutrient pool and factorial design experiments were carried out in order to identify the amino acids involved in increased biomass and induction of plasmid amplification. Leucine, glycine, and histidine were responsible for increased biomass and leucine starvation in the presence of histidine was implicated in plasmid amplification. Supercoiling of the plasmid was optimized using a dual feeding strategy. As a result of this, a fed‐batch fermentation strategy for the production of a 6.9 kb plasmid, pSVß, in Escherichia coli DH5α was developed. In batch fermentation, a maximum plasmid yield of 39.4 mg/L equivalent to 11.3 mg/g dry cell weight (DCW) was achieved with casein hydrolysate limitation. About 90% of plasmid was in the supercoiled (SC) form after 31 hr of fermentation but only remained so for a short period, leading to a very brief window for harvesting cells at scale. Subsequently, a fed‐batch fermentation using a dual feeding strategy was employed. A mean maximum plasmid yield of 44 mg/L equivalent to 9.1 mg plasmid/g DCW was achieved. After 25 hr, 90% of plasmid was in the SC form and remained at this level for the remaining 10 hr of the fermentation, allowing adequate time for the harvesting of cells without the loss of supercoiling of product. This study emphasized that optimizing fermentation strategy and identifying the essential nutrients are beneficial for bioprocessing of plasmid DNA for therapeutic applications.

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Section: Introductionmentioning

confidence: 99%

The role of amino acids in the amplification and quality of DNA vectors for industrial applications

Dorward

O’Kennedy

Folarin

et al. 2019

Biotechnology Progress

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“…Plasmids are large, covalently closed, double‐stranded DNA molecules, which are mostly found as a tightly twisted, supercoiled (sc) topoisomer and could be converted to a less compact form, the open circular (oc), by introducing a single break in one of the DNA strands . Double strand breaks will result in linear pDNA . The morphology of pDNA is very sensitive to environment conditions .…”

Section: Introductionmentioning

confidence: 99%

Plasmid production and purification: An integrated experiment‐based biochemistry and biotechnology laboratory course

Santos

Pereira

Queiroz

et al. 2019

Biochem Molecular Bio Educ

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This laboratory experiment describes the production and purification of plasmid DNA for undergraduate biochemistry and biotechnology courses. This experiment performed in a one‐week period includes the protocols for plasmid pVAX1‐LacZ production in Escherichia coli DH5α cells and subsequent purification of supercoiled pVAX1‐LacZ. Firstly, the students use a growth medium that favors the replication of the plasmid resulting in a higher plasmid production during exponential growth. Afterwards, alkaline lysis is done to disrupt the bacterial cells and recover pVAX1‐LacZ plasmid. Lastly, they perform the purification of pVAX1‐LacZ supercoiled isoform by L‐histidine chromatography, followed by agarose gel electrophoresis to characterize the separation of supercoiled isoform from contaminants. The proposed experiment provides an opportunity for students to acquire these skills that are routinely used in biochemistry and biotechnology laboratories. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):638–643, 2019.

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“…Mitochondria possess their own genome, mitochondrial DNA (mtDNA), with a gene expression system that is independent of the nuclear system, and regulate cellular functions in cooperation with the nucleus [1]. To date, many useful technologies regarding nuclear transgene expression have been reported [2][3][4][5], and a wide variety of plasmid DNA (pDNA) vectors have been developed and are now used as convenient and useful tools for transgene expression by many researchers and clinicians world-wide [5]. It is well known that mitochondria possess their own transcription/translation system and a unique codon usage that is different from universal codon usage.…”

Section: Introductionmentioning

confidence: 99%

“…Collectively, the KALA-MITO-Porter was efficiently internalized by the cells, and delivered large amounts of the pCMV-mtLuc (CGG) to mitochondria, thus contributing to the high mitochondrial transgene expression.DISCUSSIONTo achieve nuclear transgene expression, a number of useful technologies, including pDNA vectors have been reported to date[2][3][4][5]. Based on such a current trend, many researchers have focused on the mitochondrial targeting of exogenous protein via allotropic expression, where an engineered gene coding a protein fused with a mitochondrial targeting signal peptide (MTS) is transcribed/translated via nuclear transcription/cytosolic translation, and the gene product is then delivered to mitochondria via the MTS import machinery.…”

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confidence: 99%

Validation of the use of an artificial mitochondrial reporter DNA vector containing a Cytomegalovirus promoter for mitochondrial transgene expression

2017

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Mitochondria have their own gene expression system that is independent of the nuclear system, and control cellular functions in cooperation with the nucleus. While a number of useful technologies for achieving nuclear transgene expression have been reported, only a few have focused on mitochondria. In this study, we validated the utility of an artificial mitochondrial DNA vector with a virus promoter on mitochondrial transgene expression. We designed and constructed pCMV-mtLuc (CGG) that contains a CMV promotor derived from Cytomegalovirus and an artificial mitochondrial genome with a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. Nluc luciferase activity measurements showed that the pCMV-mtLuc (CGG) efficiently produced the Nluc luciferase protein in human HeLa cells. Moreover, we optimized the mitochondrial transfection of pCMV-mtLuc (CGG) using a MITO-Porter system, a liposome-based carrier for mitochondrial delivery via membrane fusion. As a result, we found that transfection of pCMV-mtLuc (CGG) by MITO-Porter modified with the KALA peptide (cationic amphipathic cell-penetrating peptide) showed a high mitochondrial transgene expression. The developed mitochondrial transgene expression system represents a potentially useful tool for the fields of nanoscience and nanotechnology for controlling the intracellular microenvironment via the regulation of mitochondrial function and promises to open additional innovative research fields of study.

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Plasmid Biopharmaceuticals

Cited by 11 publications

References 189 publications

The role of amino acids in the amplification and quality of DNA vectors for industrial applications

The role of amino acids in the amplification and quality of DNA vectors for industrial applications

Plasmid production and purification: An integrated experiment‐based biochemistry and biotechnology laboratory course

Validation of the use of an artificial mitochondrial reporter DNA vector containing a Cytomegalovirus promoter for mitochondrial transgene expression

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