Gabriel Monteiro | Instituto Superior Técnico (original) (raw)

Papers by Gabriel Monteiro

Applied microbiology and biotechnology, 2010

Structural instability has been frequently observed in natural plasmids and vectors used for prot... more Structural instability has been frequently observed in natural plasmids and vectors used for protein expression or DNA vaccine development. However, there is a lack of information concerning hotspot mapping, namely, DNA repeats or sequences identical to the host genome. This led us to evaluate the abundance and distribution of direct, inverted, and tandem repeats with high recombination potential in 36 natural plasmids from ten bacterial genera, as well as in several widely used bacterial and mammalian expression vectors. In natural plasmids, we observed an overrepresentation of close direct repeats in comparison to inverted ones and a preferential location of repeats with high recombination potential in intergenic regions, suggesting a highly plastic and dynamic behavior. In plasmid vectors, we found a high density of repeats within eukaryotic promoters and non-coding sequences. As a result of this in silico analysis, we detected a spontaneous recombination between two 21-bp direct repeats present in the human cytomegalovirus early enhancer/promoter (huCMV EEP) of the pCIneo plasmid. This finding is of particular importance, as the huCMV EEP is one of the most frequently used regulatory elements in plasmid vectors. Because pDNA integration into host gDNA can have adverse consequences in terms of plasmid processing and host safety, we also mapped several regions with high probability to mediate integration into the Escherichia coli or human genomes. Like repeated regions, some of these were located in non-coding regions of the plasmids, thus being preferential targets to be removed.

Journal of biotechnology, 2009

Plasmid pCIneo is a ColE1-like mammalian expression vector also used as backbone for DNA vaccine ... more Plasmid pCIneo is a ColE1-like mammalian expression vector also used as backbone for DNA vaccine development. We have recently shown that pCIneo spontaneously recombines due to the presence of two 28 bp direct repeats. The persistence of low-frequency recombinants led us to evaluate the impact of environmental stresses typically found during plasmid production on plasmid copy number and recombination frequency. We observed an increase in pCIneo amplification (2.6-4.3-fold) in Escherichia coli cultures grown at 42 • C and also in minimal medium (at both 37 • C and 42 • C). These conditions fit to the smallest ratio between recombinant molecules and total plasmids. Conversely, increasing the dissolved oxygen tension from 20% to 40% in rich media did not have a significant impact on both plasmid copy number and recombination frequency, independently of the temperature used.

Trends in Biotechnology, 2009

The global increase in the number of applications involving therapeutic plasmid DNA (pDNA) is cre... more The global increase in the number of applications involving therapeutic plasmid DNA (pDNA) is creating a need for large amounts of highly stable and purified molecules. One of the main obstacles during the developmental stages of a new therapeutic DNA molecule involves tackling a wide array of structural instability events occurring in/with pDNA and therefore assuring its structural integrity. This review focuses on major instability determinants in pDNA. Their elimination could be considered an important step towards the design of safer and more efficient plasmid molecules. Particular emphasis is given to mutations triggered by the presence of repeated sequences, instability events occurring during plasmid intracellular routing, instability mediated by insertion sequences and host genome integration.

Molecular biotechnology, 2008

The stability in Escherichia coli of a mammalian expression vector (pCIneo) and its derivative ca... more The stability in Escherichia coli of a mammalian expression vector (pCIneo) and its derivative candidate DNA vaccine (pGPV-PV) is described. These multicopy pMB1-type plasmids were found to recombine in several recA E. coli strains due to the presence of two 28 bp direct repeats flanking intervening sequences of 1.6 kb (pCIneo) and 3.2 kb (pGPV-PV). In this recombination event, one of the direct repeats and the intervening sequence were deleted or duplicated, originating monomeric or/and hetero-dimeric plasmid forms, respectively. Additionally, the plasmid rearrangement led to the acquisition of a kanamycin resistance phenotype. Recombination frequencies between 7.8 × 10−7 and 3.1 × 10−5 were determined for DH5α and JM109(DE3) strains, respectively. Higher recombination frequencies were found in cells previously grown up to stationary growth phase being the monomeric plasmid form the prevalent one. Real-time PCR quantification revealed the presence of approximately 1.5 × 104 recombined molecules per 2 × 105 cells pre-kanamycin exposure. Under selective pressure of this antibiotic, the number of recombined molecules increased approximately 2,000-fold in a 48-h period replacing the original plasmid form. The high frequency, at which deletion-duplication occurred in the absence of kanamycin selective pressure, should be regarded as a safety concern. This work highlights the impact of mutational hot spots on expression and cloning plasmid vectors and the need to carefully design plasmid vectors.

Mobile DNA, 2012

Background: Transposition in IS3, IS30, IS21 and IS256 insertion sequence (IS) families utilizes ... more Background: Transposition in IS3, IS30, IS21 and IS256 insertion sequence (IS) families utilizes an unconventional two-step pathway. A figure-of-eight intermediate in Step I, from asymmetric single-strand cleavage and joining reactions, is converted into a double-stranded minicircle whose junction (the abutted left and right ends) is the substrate for symmetrical transesterification attacks on target DNA in Step II, suggesting intrinsically different synaptic complexes (SC) for each step. Transposases of these ISs bind poorly to cognate DNA and comparative biophysical analyses of SC I and SC II have proven elusive. We have prepared a native, soluble, active, GFP-tagged fusion derivative of the IS2 transposase that creates fully formed complexes with single-end and minicircle junction (MCJ) substrates and used these successfully in hydroxyl radical footprinting experiments.

Journal of Controlled …, Jan 1, 2012

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene th... more The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA:LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies.

Current Bionanotechnology, 2015

Applied microbiology and biotechnology, 2014

Insertion specificity of mobile genetic elements is a rather complex aspect of DNA transposition,... more Insertion specificity of mobile genetic elements is a rather complex aspect of DNA transposition, which, despite much progress towards its elucidation, still remains incompletely understood. We report here the results of a meta-analysis of IS2 target sites from genomic, phage, and plasmid DNA and find that newly acquired IS2 elements are consistently inserted around abrupt DNA compositional shifts, particularly in the form of switch sites of GC skew. The results presented in this study not only corroborate our previous observations that both the insertion sequence (IS) minicircle junction and target region adopt intrinsically bent conformations in IS2, but most interestingly, extend this requirement to other families of IS elements. Using this information, we were able to pinpoint regions with high propensity for transposition and to predict and detect, de novo, a novel IS2 insertion event in the 3′ region of the gfp gene of a reporter plasmid. We also found that during amplification of this plasmid, process parameters such as scale, culture growth phase, and medium composition exacerbate IS2 transposition, leading to contamination levels with potentially detrimental clinical effects. Overall, our findings provide new insights into the role of target DNA structure in the mechanism of transposition of IS elements and extend our understanding of how culture conditions are a relevant factor in the induction of genetic instability.

Methods in Molecular Biology, 2016

Maedi-visna virus (MVV) is an ovine retrovirus of the Lentivirus genus, responsible for a chronic... more Maedi-visna virus (MVV) is an ovine retrovirus of the Lentivirus genus, responsible for a chronic and progressive disease of sheep with a high prevalence all over the world. Therefore, measures aiming at the control of MVV infection are necessary, and the development of DNA vaccines may be the ideal approach. A DNA vaccine is an antigen-encoding bacterial plasmid designed to mimic infections safely, with ability to generate both humoral and cellular long-lasting immune responses once it is delivered to the host.Here, we describe the development and evaluation of DNA vaccines against ovine maedi-visna virus. The first step is the design of the vaccines, including the choice of the backbone vector and the nucleotide sequences to use as antigen-encoding sequences. Once constructed, the vaccines may be produced with high quality for use in in vitro and in vivo tests. In vitro assays are performed through transfection of animal cells to confirm the expression of the protein, while in vivo tests are carried out by mouse and/or sheep immunization in order to check humoral and cellular responses to the vaccines and conclude about their efficiency. Several approaches may be later performed in order to enhance the effectiveness of the vaccines, such as the introduction of targeting sequences, the use of a prime-boost strategy, the administration of a combined vaccine, and the use of liposomes as delivery vehicle.

Journal of Chromatography A, 2016

Minicircle (MC) DNA vectors are able to generate a high-level transgene expression in vivo, which... more Minicircle (MC) DNA vectors are able to generate a high-level transgene expression in vivo, which is superior to the one afforded by conventional plasmids. MC vectors are produced by replicating a parental plasmid (PP) and promoting its recombination in Escherichia coli. This generates a MC with the expression cassette, and a miniplasmid (MP) with the replication segment. Unfortunately, wider use of MC vectors is hampered by difficulties in isolating the target MCs from their MP counterpart. In this proof-of-concept study, a reproducible process is described to improve the purification of supercoiled (sc) MCs that combines an in vitro enzymatic relaxation of sc MP impurities with topoisomer separation and RNA clearance by hydrophobic interaction chromatography (HIC) step. At the early stage of vector design, a site for the nicking endonuclease Nb.BbvCI was strategically placed in the MP part of the PP backbone. A process was then established that involves E. coli culture and recombination of PPs into target MC, cell harvesting and alkaline lysis, precipitation with isopropanol and ammonium sulfate and diafiltration/concentration by microfiltration. Next, an in vitro digestion step was carried out with Nb.BbvCI to nick of one of the strands of the MPs and of non-recombined PPs by Nb.BbvCI. As a result, sc MPs and non-recombined PPs were converted into the corresponding open circular (oc) forms whereas sc MCs remain unaffected. Finally, sc MC was isolated from oc DNA molecules (oc MPs, oc MC) and RNA by performing HIC with a phenyl-Sepharose column using a series of elution steps with decreasing ammonium sulfate concentrations. On the basis of agarose gel electrophoresis analysis, the sc MC-containing fractions were determined to be virtually free from nucleic acid impurities.

Building Pathology and Rehabilitation, 2016

Biotechnol Bioeng, 1999

Two important issues in the downstream processing of plasmids for gene therapy are the stability ... more Two important issues in the downstream processing of plasmids for gene therapy are the stability of plasmids in the process streams, and the presence of contaminating host RNA. Results with a 4.8-kb plasmid harbored in a non-nuclease-deficient strain of Escherichia coli show that, in spite of the harsh conditions during alkaline lysis, a fraction of endogenous nucleases remains active, degrading both RNA and genomic and plasmid DNA. Although it is possible to minimize plasmid degradation by decreasing temperature and reducing processing times, the presence of endogenous nucleases can be used advantageously to purify the plasmid streams. The kinetics of nucleic acid degradation showed that, by controlling the incubation at 37 degrees C, it was possible to degrade RNA selectively, while maintaining plasmid integrity. A reduction of 40% in RNA content was obtained, corresponding to a 1.5-fold increase in plasmid purity using high-performance liquid chromatography (HPLC). This strategy is simple and straightforward, and the increase in processing time and the associated plasmid loss (9%) are fully justified by the purity increase. Furthermore, the use of endogenous RNase activity is clearly advantageous over alternative procedures, such as the addition of external RNase, in terms of cost, validation, and compliance with guidelines from regulatory agencies.

Biotechnology and Bioengineering

ABSTRACT

Mobile DNA, 2012

Background: Transposition in IS3, IS30, IS21 and IS256 insertion sequence (IS) families utilizes ... more Background: Transposition in IS3, IS30, IS21 and IS256 insertion sequence (IS) families utilizes an unconventional two-step pathway. A figure-of-eight intermediate in Step I, from asymmetric single-strand cleavage and joining reactions, is converted into a double-stranded minicircle whose junction (the abutted left and right ends) is the substrate for symmetrical transesterification attacks on target DNA in Step II, suggesting intrinsically different synaptic complexes (SC) for each step. Transposases of these ISs bind poorly to cognate DNA and comparative biophysical analyses of SC I and SC II have proven elusive. We have prepared a native, soluble, active, GFP-tagged fusion derivative of the IS2 transposase that creates fully formed complexes with single-end and minicircle junction (MCJ) substrates and used these successfully in hydroxyl radical footprinting experiments.

Biotechnology Journal, 2011

As the number of applications involving therapeutic plasmid DNA (pDNA) increases worldwide, there... more As the number of applications involving therapeutic plasmid DNA (pDNA) increases worldwide, there is a growing concern over maintaining rigorous quality control through a panel of highquality assays. For this reason, efficient, cost-effective and sensitive technologies enabling the identification of genetic variants and unwanted side products are needed to successfully establish the identity and stability of a plasmid-based biopharmaceutical. This review highlights several bioinformatic tools for ab initio detection of potentially unstable DNA regions, as well as techniques used for mutation detection in nucleic acids, with particular emphasis on pDNA.

Applied microbiology and biotechnology, 2010

Structural instability has been frequently observed in natural plasmids and vectors used for prot... more Structural instability has been frequently observed in natural plasmids and vectors used for protein expression or DNA vaccine development. However, there is a lack of information concerning hotspot mapping, namely, DNA repeats or sequences identical to the host genome. This led us to evaluate the abundance and distribution of direct, inverted, and tandem repeats with high recombination potential in 36 natural plasmids from ten bacterial genera, as well as in several widely used bacterial and mammalian expression vectors. In natural plasmids, we observed an overrepresentation of close direct repeats in comparison to inverted ones and a preferential location of repeats with high recombination potential in intergenic regions, suggesting a highly plastic and dynamic behavior. In plasmid vectors, we found a high density of repeats within eukaryotic promoters and non-coding sequences. As a result of this in silico analysis, we detected a spontaneous recombination between two 21-bp direct repeats present in the human cytomegalovirus early enhancer/promoter (huCMV EEP) of the pCIneo plasmid. This finding is of particular importance, as the huCMV EEP is one of the most frequently used regulatory elements in plasmid vectors. Because pDNA integration into host gDNA can have adverse consequences in terms of plasmid processing and host safety, we also mapped several regions with high probability to mediate integration into the Escherichia coli or human genomes. Like repeated regions, some of these were located in non-coding regions of the plasmids, thus being preferential targets to be removed.

Journal of biotechnology, 2009

Plasmid pCIneo is a ColE1-like mammalian expression vector also used as backbone for DNA vaccine ... more Plasmid pCIneo is a ColE1-like mammalian expression vector also used as backbone for DNA vaccine development. We have recently shown that pCIneo spontaneously recombines due to the presence of two 28 bp direct repeats. The persistence of low-frequency recombinants led us to evaluate the impact of environmental stresses typically found during plasmid production on plasmid copy number and recombination frequency. We observed an increase in pCIneo amplification (2.6-4.3-fold) in Escherichia coli cultures grown at 42 • C and also in minimal medium (at both 37 • C and 42 • C). These conditions fit to the smallest ratio between recombinant molecules and total plasmids. Conversely, increasing the dissolved oxygen tension from 20% to 40% in rich media did not have a significant impact on both plasmid copy number and recombination frequency, independently of the temperature used.

Trends in Biotechnology, 2009

The global increase in the number of applications involving therapeutic plasmid DNA (pDNA) is cre... more The global increase in the number of applications involving therapeutic plasmid DNA (pDNA) is creating a need for large amounts of highly stable and purified molecules. One of the main obstacles during the developmental stages of a new therapeutic DNA molecule involves tackling a wide array of structural instability events occurring in/with pDNA and therefore assuring its structural integrity. This review focuses on major instability determinants in pDNA. Their elimination could be considered an important step towards the design of safer and more efficient plasmid molecules. Particular emphasis is given to mutations triggered by the presence of repeated sequences, instability events occurring during plasmid intracellular routing, instability mediated by insertion sequences and host genome integration.

Molecular biotechnology, 2008

The stability in Escherichia coli of a mammalian expression vector (pCIneo) and its derivative ca... more The stability in Escherichia coli of a mammalian expression vector (pCIneo) and its derivative candidate DNA vaccine (pGPV-PV) is described. These multicopy pMB1-type plasmids were found to recombine in several recA E. coli strains due to the presence of two 28 bp direct repeats flanking intervening sequences of 1.6 kb (pCIneo) and 3.2 kb (pGPV-PV). In this recombination event, one of the direct repeats and the intervening sequence were deleted or duplicated, originating monomeric or/and hetero-dimeric plasmid forms, respectively. Additionally, the plasmid rearrangement led to the acquisition of a kanamycin resistance phenotype. Recombination frequencies between 7.8 × 10−7 and 3.1 × 10−5 were determined for DH5α and JM109(DE3) strains, respectively. Higher recombination frequencies were found in cells previously grown up to stationary growth phase being the monomeric plasmid form the prevalent one. Real-time PCR quantification revealed the presence of approximately 1.5 × 104 recombined molecules per 2 × 105 cells pre-kanamycin exposure. Under selective pressure of this antibiotic, the number of recombined molecules increased approximately 2,000-fold in a 48-h period replacing the original plasmid form. The high frequency, at which deletion-duplication occurred in the absence of kanamycin selective pressure, should be regarded as a safety concern. This work highlights the impact of mutational hot spots on expression and cloning plasmid vectors and the need to carefully design plasmid vectors.

Mobile DNA, 2012

Background: Transposition in IS3, IS30, IS21 and IS256 insertion sequence (IS) families utilizes ... more Background: Transposition in IS3, IS30, IS21 and IS256 insertion sequence (IS) families utilizes an unconventional two-step pathway. A figure-of-eight intermediate in Step I, from asymmetric single-strand cleavage and joining reactions, is converted into a double-stranded minicircle whose junction (the abutted left and right ends) is the substrate for symmetrical transesterification attacks on target DNA in Step II, suggesting intrinsically different synaptic complexes (SC) for each step. Transposases of these ISs bind poorly to cognate DNA and comparative biophysical analyses of SC I and SC II have proven elusive. We have prepared a native, soluble, active, GFP-tagged fusion derivative of the IS2 transposase that creates fully formed complexes with single-end and minicircle junction (MCJ) substrates and used these successfully in hydroxyl radical footprinting experiments.

Journal of Controlled …, Jan 1, 2012

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene th... more The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA:LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies.

Current Bionanotechnology, 2015

Applied microbiology and biotechnology, 2014

Insertion specificity of mobile genetic elements is a rather complex aspect of DNA transposition,... more Insertion specificity of mobile genetic elements is a rather complex aspect of DNA transposition, which, despite much progress towards its elucidation, still remains incompletely understood. We report here the results of a meta-analysis of IS2 target sites from genomic, phage, and plasmid DNA and find that newly acquired IS2 elements are consistently inserted around abrupt DNA compositional shifts, particularly in the form of switch sites of GC skew. The results presented in this study not only corroborate our previous observations that both the insertion sequence (IS) minicircle junction and target region adopt intrinsically bent conformations in IS2, but most interestingly, extend this requirement to other families of IS elements. Using this information, we were able to pinpoint regions with high propensity for transposition and to predict and detect, de novo, a novel IS2 insertion event in the 3′ region of the gfp gene of a reporter plasmid. We also found that during amplification of this plasmid, process parameters such as scale, culture growth phase, and medium composition exacerbate IS2 transposition, leading to contamination levels with potentially detrimental clinical effects. Overall, our findings provide new insights into the role of target DNA structure in the mechanism of transposition of IS elements and extend our understanding of how culture conditions are a relevant factor in the induction of genetic instability.

Methods in Molecular Biology, 2016

Maedi-visna virus (MVV) is an ovine retrovirus of the Lentivirus genus, responsible for a chronic... more Maedi-visna virus (MVV) is an ovine retrovirus of the Lentivirus genus, responsible for a chronic and progressive disease of sheep with a high prevalence all over the world. Therefore, measures aiming at the control of MVV infection are necessary, and the development of DNA vaccines may be the ideal approach. A DNA vaccine is an antigen-encoding bacterial plasmid designed to mimic infections safely, with ability to generate both humoral and cellular long-lasting immune responses once it is delivered to the host.Here, we describe the development and evaluation of DNA vaccines against ovine maedi-visna virus. The first step is the design of the vaccines, including the choice of the backbone vector and the nucleotide sequences to use as antigen-encoding sequences. Once constructed, the vaccines may be produced with high quality for use in in vitro and in vivo tests. In vitro assays are performed through transfection of animal cells to confirm the expression of the protein, while in vivo tests are carried out by mouse and/or sheep immunization in order to check humoral and cellular responses to the vaccines and conclude about their efficiency. Several approaches may be later performed in order to enhance the effectiveness of the vaccines, such as the introduction of targeting sequences, the use of a prime-boost strategy, the administration of a combined vaccine, and the use of liposomes as delivery vehicle.

Journal of Chromatography A, 2016

Minicircle (MC) DNA vectors are able to generate a high-level transgene expression in vivo, which... more Minicircle (MC) DNA vectors are able to generate a high-level transgene expression in vivo, which is superior to the one afforded by conventional plasmids. MC vectors are produced by replicating a parental plasmid (PP) and promoting its recombination in Escherichia coli. This generates a MC with the expression cassette, and a miniplasmid (MP) with the replication segment. Unfortunately, wider use of MC vectors is hampered by difficulties in isolating the target MCs from their MP counterpart. In this proof-of-concept study, a reproducible process is described to improve the purification of supercoiled (sc) MCs that combines an in vitro enzymatic relaxation of sc MP impurities with topoisomer separation and RNA clearance by hydrophobic interaction chromatography (HIC) step. At the early stage of vector design, a site for the nicking endonuclease Nb.BbvCI was strategically placed in the MP part of the PP backbone. A process was then established that involves E. coli culture and recombination of PPs into target MC, cell harvesting and alkaline lysis, precipitation with isopropanol and ammonium sulfate and diafiltration/concentration by microfiltration. Next, an in vitro digestion step was carried out with Nb.BbvCI to nick of one of the strands of the MPs and of non-recombined PPs by Nb.BbvCI. As a result, sc MPs and non-recombined PPs were converted into the corresponding open circular (oc) forms whereas sc MCs remain unaffected. Finally, sc MC was isolated from oc DNA molecules (oc MPs, oc MC) and RNA by performing HIC with a phenyl-Sepharose column using a series of elution steps with decreasing ammonium sulfate concentrations. On the basis of agarose gel electrophoresis analysis, the sc MC-containing fractions were determined to be virtually free from nucleic acid impurities.

Building Pathology and Rehabilitation, 2016

Biotechnol Bioeng, 1999

Two important issues in the downstream processing of plasmids for gene therapy are the stability ... more Two important issues in the downstream processing of plasmids for gene therapy are the stability of plasmids in the process streams, and the presence of contaminating host RNA. Results with a 4.8-kb plasmid harbored in a non-nuclease-deficient strain of Escherichia coli show that, in spite of the harsh conditions during alkaline lysis, a fraction of endogenous nucleases remains active, degrading both RNA and genomic and plasmid DNA. Although it is possible to minimize plasmid degradation by decreasing temperature and reducing processing times, the presence of endogenous nucleases can be used advantageously to purify the plasmid streams. The kinetics of nucleic acid degradation showed that, by controlling the incubation at 37 degrees C, it was possible to degrade RNA selectively, while maintaining plasmid integrity. A reduction of 40% in RNA content was obtained, corresponding to a 1.5-fold increase in plasmid purity using high-performance liquid chromatography (HPLC). This strategy is simple and straightforward, and the increase in processing time and the associated plasmid loss (9%) are fully justified by the purity increase. Furthermore, the use of endogenous RNase activity is clearly advantageous over alternative procedures, such as the addition of external RNase, in terms of cost, validation, and compliance with guidelines from regulatory agencies.

Biotechnology and Bioengineering

ABSTRACT

Mobile DNA, 2012

Background: Transposition in IS3, IS30, IS21 and IS256 insertion sequence (IS) families utilizes ... more Background: Transposition in IS3, IS30, IS21 and IS256 insertion sequence (IS) families utilizes an unconventional two-step pathway. A figure-of-eight intermediate in Step I, from asymmetric single-strand cleavage and joining reactions, is converted into a double-stranded minicircle whose junction (the abutted left and right ends) is the substrate for symmetrical transesterification attacks on target DNA in Step II, suggesting intrinsically different synaptic complexes (SC) for each step. Transposases of these ISs bind poorly to cognate DNA and comparative biophysical analyses of SC I and SC II have proven elusive. We have prepared a native, soluble, active, GFP-tagged fusion derivative of the IS2 transposase that creates fully formed complexes with single-end and minicircle junction (MCJ) substrates and used these successfully in hydroxyl radical footprinting experiments.

Biotechnology Journal, 2011

As the number of applications involving therapeutic plasmid DNA (pDNA) increases worldwide, there... more As the number of applications involving therapeutic plasmid DNA (pDNA) increases worldwide, there is a growing concern over maintaining rigorous quality control through a panel of highquality assays. For this reason, efficient, cost-effective and sensitive technologies enabling the identification of genetic variants and unwanted side products are needed to successfully establish the identity and stability of a plasmid-based biopharmaceutical. This review highlights several bioinformatic tools for ab initio detection of potentially unstable DNA regions, as well as techniques used for mutation detection in nucleic acids, with particular emphasis on pDNA.