Ana Gabriela Gomes - Academia.edu (original) (raw)

Uploads

Papers by Ana Gabriela Gomes

Research paper thumbnail of Partitioning of human antibodies in polyethylene glycol–sodium citrate aqueous two-phase systems

Separation and Purification Technology, 2009

Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) an... more Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) and citrate aqueous two-phase systems (ATPSs). Initial studies performed with pure IgG solutions showed that increasing the concentration of a neutral salt, such as NaCl up to 15% (w/w), increased the partition coefficient of IgG from 0.14 to 5.21. This partitioning behaviour was also observed first in the presence of a simulated protein mixture, containing albumin and myoglobin, and then in a hybridoma cell culture supernatant. Thus, by changing the concentration of NaCl, it is possible to target the partition of IgG to the phase with fewer impurities. The partial purification of IgG from a hybridoma cell culture supernatant in a serum-containing media was achieved using 15% NaCl and recovering the antibody in the top phase. Using an ATPS composed of 8% (w/w) PEG 3350, 8% (w/w) citrate and 15% (w/w) NaCl at pH 6, IgG was recovered with a 99% yield, 44% HPLC purity and with a IgG/protein ratio of 0.9. IgG can be re-extracted to a new citrate phase by decreasing the overall concentration of NaCl to 5%. This back-extraction step introduces not only a further purification step but also allows the separation of the antibody from the polymer. IgG was recovered with a global yield of 99%, with a HPLC purity of 76% and with a IgG/protein ratio of 0.96.

Research paper thumbnail of Evidence that the insertion events of IS2 transposition are biased towards abrupt compositional shifts in target DNA and modulated by a diverse set of culture parameters

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 amplific...

Research paper thumbnail of Purification of plasmid DNA with aqueous two phase systems of PEG 600 and sodium citrate/ammonium sulfate

Separation and Purification Technology, 2009

Polymer/salt aqueous two phase systems (ATPS) based on polyethylene glycol (PEG) 600, sodium citr... more Polymer/salt aqueous two phase systems (ATPS) based on polyethylene glycol (PEG) 600, sodium citrate and ammonium sulfate were used to partially purify plasmid DNA (pDNA) from Escherichia coli alkaline lysates. The effect of pH and lysate load on the binodal curve was analyzed and tie-lines were determined in order to establish the optimal conditions for ATPS formation. A series of extraction experiments were performed at pH 6.9 using a 20% (w/w) lysate load and systems with 16.5% (w/w) salt, 19.0% (w/w) PEG 600, and a tie-line length of 37.1% (w/w). Under these conditions, plasmid DNA was recovered in the salt-rich bottom phase. However, whereas with sodium citrate-based systems recovery yields closer to 100% were obtained, the use of ammonium sulfate enabled higher purification although with lower recoveries. Thus, the performance of ATPS prepared by combining sodium citrate and ammonium sulfate was also evaluated. A mixture of 25% (w/w) ammonium sulfate and 75% (w/w) sodium citrate offered a good compromise between plasmid recovery (91.1%) and purity (17.2%). Multi-step extraction procedures were evaluated in order to improve the process performance. Although the majority of the impurities were removed in the first step, incremental increases in the purity were obtained with the inclusion of extra steps. The top to bottom phase volume ratio was increased in order to increase plasmid concentration in the bottom phase. Although this was achieved using a phase ratio of 4, it was not possible to concentrate plasmid relatively to the starting lysate. Overall, the results show that ammonium sulfate, a salt which has a high environmental impact, can be partially replaced in ATPS by sodium citrate, without significant decrease in the performance of plasmid purification.

Research paper thumbnail of Comparative Transcriptomics of Arabidopsis Sperm Cells  

Plant Physiology, 2008

In flowering plants, the two sperm cells are embedded within the cytoplasm of the growing pollen ... more In flowering plants, the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part in fertilization are crucial goals in the study of plant reproduction. Studies of gene expression in male gametes of maize (Zea mays) and Plumbago and in lily (Lilium longiflorum) generative cells already showed that the previously held view of transcriptionally inert male gametes was not true, but genome-wide studies were lacking. Analyses in the model plant Arabidopsis (Arabidopsis thaliana) were hindered, because no method to isolate sperm cells was available. Here, we used fluorescence-activated cell sorting to isolate sperm cells from Arabidopsis, allowing GeneChip analysis of their transcriptome at a genome-wide level. Comparative analysis of the sperm cell transcriptome with thos...

Research paper thumbnail of Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography

Journal of Molecular Recognition, 2010

Research paper thumbnail of Studies on the adsorption of cell impurities from plasmid-containing lysates to phenyl boronic acid chromatographic beads

Journal of Chromatography A, 2011

Plasmid DNA (pDNA) is purified directly from alkaline lysis-derived Escherichia coli (E. coli) ly... more Plasmid DNA (pDNA) is purified directly from alkaline lysis-derived Escherichia coli (E. coli) lysates by phenyl boronate (PB) chromatography. The method explores the ability of PB ligands to bind covalently, but reversibly, to cis-diol-containing impurities like RNA and lipopolysaccharides (LPS), leaving pDNA in solution. In spite of this specificity, cis-diol free species like proteins and genomic DNA (gDNA) are also removed. This is a major advantage since the process is designed to keep the target pDNA from binding. The focus of this paper is on the study of the secondary interactions between the impurities (RNA, gDNA, proteins, LPS) in a pDNA-containing lysate and 3-amino PB controlled pore glass (CPG) matrices. Runs were designed to evaluate the role of adsorption buffer composition, feed type (pH, salt content), CPG matrix and sample pretreatment (RNase A, isopropanol precipitation). Water was chosen as the adsorption buffer over MgCl 2 solutions since it maximised pDNA yield (96.2 ± 4.9%) and protein removal (61.3 ± 3.0%), while providing for a substantial removal of RNA (65.5 ± 3.5%) and gDNA (44.7 ± 14.1%). Although the use of pH 3.5 maximised removal of impurities (∼75%), the best compromise between plasmid yield (∼96%) and RNA clearance (∼60-70%) was obtained for a pH of 5.2. Plasmid yield was maximal (>96%) when the concentration of acetate and potassium ions in the incoming lysate feed were 1.7 M and 1.0 M, respectively. The pre-treatment of lysates with RNase A deteriorated the performance since the resulting oligoribonucleotides lack the cis-diol group at their 3 termini. Overall, the results support the idea that charge transfer interactions between the boron atom at acidic pH and electron donor groups in the aromatic bases of nucleic acids and side residues of proteins are responsible for the non-specific removal of gDNA, RNA and proteins.

Research paper thumbnail of Clearance of host cell impurities from plasmid-containing lysates by boronate adsorption

Journal of Chromatography A, 2010

The ability of boronate adsorption to clear Escherichia coli impurities directly from plasmid-con... more The ability of boronate adsorption to clear Escherichia coli impurities directly from plasmid-containing lysates (∼pH 5.2) was evaluated. Results show that 3-aminophenyl boronate (PB) controlled pore glass (CPG) is able to adsorb not only those species that bear cis-diol groups (RNA, lipopolysaccharides-LPS), and are thus able to form covalent bonds with boronate, but also cis-diol-free proteins and genomic DNA (gDNA) fragments, while leaving most plasmid DNA in solution. Control runs performed with phenyl Sepharose and with PB-free CPG beads ruled out hydrophobic interactions with the phenyl ring and non-specific interactions with the glass matrix, respectively, as being responsible for RNA and gDNA adsorption. In batch mode, up to 97.6 ± 3.1% of RNA, 94.6 ± 0.8% of proteins and 96.7 ± 11.7% of gDNA were cleared after 30 min, with a plasmid yield of 64%. In fixed-bed mode, most of the plasmid was recovered in the flowthrough (96.2 ± 4.0%), even though the RNA (65.5 ± 2.8%), protein (84.4 ± 1.3%) and gDNA clearance (44.7 ± 14.1%) were not as effective. In both cases, the LPS content was removed to a residual value of less than 0.005 EU/ml. The method is fast and straightforward, circumvents the need for pre-treatment of the feed and may contribute to shorten plasmid purification processes, as the treated streams can proceed directly to the final polishing steps.

Research paper thumbnail of Partitioning of human antibodies in polyethylene glycol–sodium citrate aqueous two-phase systems

Separation and Purification Technology, 2009

Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) an... more Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) and citrate aqueous two-phase systems (ATPSs). Initial studies performed with pure IgG solutions showed that increasing the concentration of a neutral salt, such as NaCl up to 15% (w/w), increased the partition coefficient of IgG from 0.14 to 5.21. This partitioning behaviour was also observed first in the presence of a simulated protein mixture, containing albumin and myoglobin, and then in a hybridoma cell culture supernatant. Thus, by changing the concentration of NaCl, it is possible to target the partition of IgG to the phase with fewer impurities. The partial purification of IgG from a hybridoma cell culture supernatant in a serum-containing media was achieved using 15% NaCl and recovering the antibody in the top phase. Using an ATPS composed of 8% (w/w) PEG 3350, 8% (w/w) citrate and 15% (w/w) NaCl at pH 6, IgG was recovered with a 99% yield, 44% HPLC purity and with a IgG/protein ratio of 0.9. IgG can be re-extracted to a new citrate phase by decreasing the overall concentration of NaCl to 5%. This back-extraction step introduces not only a further purification step but also allows the separation of the antibody from the polymer. IgG was recovered with a global yield of 99%, with a HPLC purity of 76% and with a IgG/protein ratio of 0.96.

Research paper thumbnail of Evidence that the insertion events of IS2 transposition are biased towards abrupt compositional shifts in target DNA and modulated by a diverse set of culture parameters

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 amplific...

Research paper thumbnail of Purification of plasmid DNA with aqueous two phase systems of PEG 600 and sodium citrate/ammonium sulfate

Separation and Purification Technology, 2009

Polymer/salt aqueous two phase systems (ATPS) based on polyethylene glycol (PEG) 600, sodium citr... more Polymer/salt aqueous two phase systems (ATPS) based on polyethylene glycol (PEG) 600, sodium citrate and ammonium sulfate were used to partially purify plasmid DNA (pDNA) from Escherichia coli alkaline lysates. The effect of pH and lysate load on the binodal curve was analyzed and tie-lines were determined in order to establish the optimal conditions for ATPS formation. A series of extraction experiments were performed at pH 6.9 using a 20% (w/w) lysate load and systems with 16.5% (w/w) salt, 19.0% (w/w) PEG 600, and a tie-line length of 37.1% (w/w). Under these conditions, plasmid DNA was recovered in the salt-rich bottom phase. However, whereas with sodium citrate-based systems recovery yields closer to 100% were obtained, the use of ammonium sulfate enabled higher purification although with lower recoveries. Thus, the performance of ATPS prepared by combining sodium citrate and ammonium sulfate was also evaluated. A mixture of 25% (w/w) ammonium sulfate and 75% (w/w) sodium citrate offered a good compromise between plasmid recovery (91.1%) and purity (17.2%). Multi-step extraction procedures were evaluated in order to improve the process performance. Although the majority of the impurities were removed in the first step, incremental increases in the purity were obtained with the inclusion of extra steps. The top to bottom phase volume ratio was increased in order to increase plasmid concentration in the bottom phase. Although this was achieved using a phase ratio of 4, it was not possible to concentrate plasmid relatively to the starting lysate. Overall, the results show that ammonium sulfate, a salt which has a high environmental impact, can be partially replaced in ATPS by sodium citrate, without significant decrease in the performance of plasmid purification.

Research paper thumbnail of Comparative Transcriptomics of Arabidopsis Sperm Cells  

Plant Physiology, 2008

In flowering plants, the two sperm cells are embedded within the cytoplasm of the growing pollen ... more In flowering plants, the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part in fertilization are crucial goals in the study of plant reproduction. Studies of gene expression in male gametes of maize (Zea mays) and Plumbago and in lily (Lilium longiflorum) generative cells already showed that the previously held view of transcriptionally inert male gametes was not true, but genome-wide studies were lacking. Analyses in the model plant Arabidopsis (Arabidopsis thaliana) were hindered, because no method to isolate sperm cells was available. Here, we used fluorescence-activated cell sorting to isolate sperm cells from Arabidopsis, allowing GeneChip analysis of their transcriptome at a genome-wide level. Comparative analysis of the sperm cell transcriptome with thos...

Research paper thumbnail of Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography

Journal of Molecular Recognition, 2010

Research paper thumbnail of Studies on the adsorption of cell impurities from plasmid-containing lysates to phenyl boronic acid chromatographic beads

Journal of Chromatography A, 2011

Plasmid DNA (pDNA) is purified directly from alkaline lysis-derived Escherichia coli (E. coli) ly... more Plasmid DNA (pDNA) is purified directly from alkaline lysis-derived Escherichia coli (E. coli) lysates by phenyl boronate (PB) chromatography. The method explores the ability of PB ligands to bind covalently, but reversibly, to cis-diol-containing impurities like RNA and lipopolysaccharides (LPS), leaving pDNA in solution. In spite of this specificity, cis-diol free species like proteins and genomic DNA (gDNA) are also removed. This is a major advantage since the process is designed to keep the target pDNA from binding. The focus of this paper is on the study of the secondary interactions between the impurities (RNA, gDNA, proteins, LPS) in a pDNA-containing lysate and 3-amino PB controlled pore glass (CPG) matrices. Runs were designed to evaluate the role of adsorption buffer composition, feed type (pH, salt content), CPG matrix and sample pretreatment (RNase A, isopropanol precipitation). Water was chosen as the adsorption buffer over MgCl 2 solutions since it maximised pDNA yield (96.2 ± 4.9%) and protein removal (61.3 ± 3.0%), while providing for a substantial removal of RNA (65.5 ± 3.5%) and gDNA (44.7 ± 14.1%). Although the use of pH 3.5 maximised removal of impurities (∼75%), the best compromise between plasmid yield (∼96%) and RNA clearance (∼60-70%) was obtained for a pH of 5.2. Plasmid yield was maximal (>96%) when the concentration of acetate and potassium ions in the incoming lysate feed were 1.7 M and 1.0 M, respectively. The pre-treatment of lysates with RNase A deteriorated the performance since the resulting oligoribonucleotides lack the cis-diol group at their 3 termini. Overall, the results support the idea that charge transfer interactions between the boron atom at acidic pH and electron donor groups in the aromatic bases of nucleic acids and side residues of proteins are responsible for the non-specific removal of gDNA, RNA and proteins.

Research paper thumbnail of Clearance of host cell impurities from plasmid-containing lysates by boronate adsorption

Journal of Chromatography A, 2010

The ability of boronate adsorption to clear Escherichia coli impurities directly from plasmid-con... more The ability of boronate adsorption to clear Escherichia coli impurities directly from plasmid-containing lysates (∼pH 5.2) was evaluated. Results show that 3-aminophenyl boronate (PB) controlled pore glass (CPG) is able to adsorb not only those species that bear cis-diol groups (RNA, lipopolysaccharides-LPS), and are thus able to form covalent bonds with boronate, but also cis-diol-free proteins and genomic DNA (gDNA) fragments, while leaving most plasmid DNA in solution. Control runs performed with phenyl Sepharose and with PB-free CPG beads ruled out hydrophobic interactions with the phenyl ring and non-specific interactions with the glass matrix, respectively, as being responsible for RNA and gDNA adsorption. In batch mode, up to 97.6 ± 3.1% of RNA, 94.6 ± 0.8% of proteins and 96.7 ± 11.7% of gDNA were cleared after 30 min, with a plasmid yield of 64%. In fixed-bed mode, most of the plasmid was recovered in the flowthrough (96.2 ± 4.0%), even though the RNA (65.5 ± 2.8%), protein (84.4 ± 1.3%) and gDNA clearance (44.7 ± 14.1%) were not as effective. In both cases, the LPS content was removed to a residual value of less than 0.005 EU/ml. The method is fast and straightforward, circumvents the need for pre-treatment of the feed and may contribute to shorten plasmid purification processes, as the treated streams can proceed directly to the final polishing steps.