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The Effect of Recombinant Protein Production in Lactococcus lactis Transcriptome and Proteome

The Effect of Recombinant Protein Production in Lactococcus lactis Transcriptome and Proteome | iBB | Scoop.it

Lactococcus lactis is a food-grade, and generally recognized as safe, bacterium, which making it ideal for producing plasmid DNA (pDNA) or recombinant proteins for industrial or pharmaceutical applications. A paper published in Microorganisms by Sofia Duarte and Gabriel Monteiro from BERG-iBB reviews the major findings from L. lactis transcriptome and proteome studies, with an overexpression of native or recombinant proteins. These studies provide important insights on how to engineer the plasmid vectors and/or the strains in order to achieve high pDNA or recombinant proteins yields, with high quality standards. 

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Cláudia Alves to Defend PhD Thesis in Biotechnology and Biosciences

Cláudia Alves to Defend PhD Thesis in Biotechnology and Biosciences | iBB | Scoop.it

Cláudia Patrícia Almeida Alves will be defending her PhD thesis in Biotechnology and Biosciences at Instituto Superior Técnico, Friday the 14th of May 2021, at 14:00 am (https://videoconf-colibri.zoom.us/j/82471896554). During the last years, and under the supervision of Gabriel Monteiro and Miguel Prazeres from iBB, Cláudia engineered Escherichia coli strains for the production of minicircle vectors by resolvase-catalyzed in vivo recombination. The title of her thesis is “Escherichia coli Genome Engineering For Improved Minicircle Production". Yo can check more about Cláudia's work here.

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Plasmid Replicons for the Production of Biopharmaceuticals by Lactococcus lactis Cell Factories

Plasmid Replicons for the Production of Biopharmaceuticals by Lactococcus lactis Cell Factories | iBB | Scoop.it

While Lactococcus lactis is traditionally associated with the fermented food industry, applications of these bacteria have been spreading to the pharmaceutical industry. The goal is to use L. lactis as cell factories for the production of added-value recombinant proteins and plasmid DNA (pDNA) for DNA vaccination, as a safer and industrially profitable alternative to the traditional Escherichia coli host. In a recent review in International Molecular Sciences, Sofia Duarte and Gabriel Monteiro from BERG-iBB critically systematize the plasmid replicons available for the production of pharmaceutical-grade pDNA and recombinant proteins by L. lactis. The work is funded by the LactoSynt project.

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Conditioned Medium from Azurin-expressing MSC Demonstrates Anti-tumor Activity

Conditioned Medium from Azurin-expressing MSC Demonstrates Anti-tumor Activity | iBB | Scoop.it

Cell-based therapies can enhance the specificity of anti-cancer therapeutic agents. In this context, human mesenchymal stromal cells (MSC) hold a promising future as cell delivery systems for anti-cancer proteins due to their innate tropism for tumors. iBB researchers Marília Silva, Gabriel Monteiro, Arsénio Fialho, Nuno Bernardes and Cláudia Lobato da Silva, engineered human MSC through non-viral methods to secrete a human codon-optimized version of azurin (hazu), a bacterial protein with demonstrated anti-cancer activity towards different cancer models in vitro and in vivo. Upon treatment with conditioned media (CM) from these engineered cells, a decrease in cancer cell proliferation, migration and invasion was seen, and an increase in cell death was observed for breast and lung cancer cell models. The results achieved by SCERG- and BSRG-iBB researchers were published in Frontiers in Cell and Developmental Biology, Stem Cell Research section.

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Cover of Biotechnology Journal Highlights iBB's Work on Lactic Acid Bacteria

Cover of Biotechnology Journal Highlights iBB's Work on Lactic Acid Bacteria | iBB | Scoop.it

BERG-iBB researchers led by Gabriel Monteiro are engineering lactic acid bacteria and plasmid vectors in order to develop a flexible platform for biomolecule production. The cover image of the August issue of Biotechnology Journal highlights a recent contribution of the group. The work is part of the FCT-funded project LactoSynt.

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Sofia Duarte Joins LactoSynt Team as Research Scientist

Sofia Duarte Joins LactoSynt Team as Research Scientist | iBB | Scoop.it

Sofia Duarte has joined the team of the FCT-funded project "LactoSynt: Lactic Acid Bacteria as Cell Factories: a Synthetic Biology Approach for Plasmid DNA and Recombinant Protein Production". During the next three years Sofia will be responsible for engineering lactic acid bacteria and plasmid vectors with the goal of developing a flexible platform for biomolecule production. The project is a joint collaboration between iBB groups BERG (Gabriel Monteiro) and BSRG (Leonilde Moreira).

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Engineering Human MSC with VEGF-encoding Minicircles for Angiogenic Gene Therapy

Engineering Human MSC with VEGF-encoding Minicircles for Angiogenic Gene Therapy | iBB | Scoop.it

Peripheral artery disease (PAD) is a debilitating condition characterized by the blockage of arteries, which leads to limb amputation in more severe cases. Researchers from SCERG- and BERG-iBB propose the use of human bone marrow (BM) MSC transiently transfected with minicircles encoding for VEGF as an ex vivo gene therapy strategy to enhance angiogenesis in PAD patients. The data shows that VEGF overexpression improved the angiogenic potential of MSC in vitro, as confirmed by endothelial cell tube formation and cell migration assays.These results suggest that minicircle-mediated VEGF gene delivery, combined with the unique properties of human MSC, could represent a promising ex vivo gene therapy approach to improve angiogenesis in the context of PAD. The work was published in Human Gene Therapy.

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Re-engineering E. coli for Interferon Production

Re-engineering E. coli for Interferon Production | iBB | Scoop.it

The bacterial genome can be modified to favour the synthesis of recombinant protein. Such productivity gains can contribute to lower costs and increase the affordabiliy of protein pharmaceuticals. BERG-iBB researchers and colleagues from the Institute of Technology Guwahati (India) have shown that deletion of the pgi (phosphoglucose isomerase) gene in Escherichia coli favours the production of the pharmaceutically important protein interferon gamma (IFN-gamma). Specific IFN-gamma yields were 3.0-fold higher in mutant compared to native strain. Furthermore, a metabolic model analysis showed that pgi deletion raises flux efficiency towards IFN-gamma synthesis. The work was published in Enzyme Microbial Technology.

 

Photo details: 3D model of pgi by Emw, own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=8814547

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Effects of Clay's Chemical Interactions on Biocementation

Effects of Clay's Chemical Interactions on Biocementation | iBB | Scoop.it

Biocementation, or microbially induced calcite precipitation (MICP), is a technique currently being appraised for the improvement of sandy soils. A recent study led by Gabriel Monteiro from BERG-iBB and Rafaela Cardoso from IST describes an investigation of the effect of clay chemical interactions on biocementation with Sporosarcina pasteurii. Results show that osmotic effects play a key role in biocimentation and that MICP treatment in clayey soils is more complex than in sands with reduced clay content. Click on title to learn more about the research published in Applied Clay Science.

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DNA Vaccines for Aquaculture – Plasmid Delivery of Antigenic Peptides from Aeromonas hydrophila

DNA Vaccines for Aquaculture – Plasmid Delivery of Antigenic Peptides from Aeromonas hydrophila | iBB | Scoop.it
Declining wild fish stocks foster aquaculture industry for seafood supply. Such intensive fish rearing expedites disease transmission between individuals but vaccination can avoid feeding antibiotics to fish and economic losses. Plasmid DNA biopharmaceuticals research is an important BERG-iBB activity and fish vaccines development a challenge. Gabriel Monteiro, Marília Mateus and researchers from NIT-Rourkela, collaborated in designing, producing and characterizing vaccine candidates for immunizing fish against aeromoniases. DNA plasmids for expression of antigenic Aha1 peptides from A.hydrophila and a DNA nanoparticle-based delivery system were created/characterized. CHO cells transient transfection of the vaccine candidates was implemented, PLGA-chitosan nanoparticles ability for DNA complexation and protection from nucleases evaluated, and the system adequacy for up-scaled production discussed. Click on title to learn more.
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Silica Nanoparticles for Gene Therapy

Silica Nanoparticles for Gene Therapy | iBB | Scoop.it
Alternative gene delivery vehicles to augment the efficiency of transfection both in vitro and in vivo are needed. A collaborative team of researchers from iBB-BERG (Gabriel Monteiro), Centro de Química Estrutural-IST (M.C. Gonçalves) and CESAM, Universidade de Aveiro (I. Domingues) have recently described the optimization of a method to synthesize amino-functionalized silica nanoparticles. These nanparticles were complexed with plasmid DNA to assemble specialized complexes (ORMOPLEXES), which were then characterized by physical and chemical analyses and tested in vitro and in vivo for biological activity. Click on title to read the publication in Materials Science and Engineering: C.

Photo details:  TEM images of ORMOPLEXES.
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Purification of Minicircles by a Nicking Endonuclease-Assisted Method

Purification of Minicircles by a Nicking Endonuclease-Assisted Method | iBB | Scoop.it

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. Unfortunately, wider use of MC vectors is hampered by difficulties in isolating the target MCs from miniplasmid (MP) impurities. In a proof-of-concept study published in Journal of Chromatography A, a team led by Miguel Prazeres from iBB-BERG describe a reproducible process to improve the purification of supercoiled (sc) MCs. The study shows that sc MC can be obtained completely free from nucleic acid impurities by modifying the topology of MP impurities with a nicking endonuclease and subsequently exploring the ability of hydrophonic interaction chromatography to fractionate topoisomers.

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Salomé Magalhães Defends PhD Thesis in Biotechnology

Salomé Magalhães Defends PhD Thesis in Biotechnology | iBB | Scoop.it

Salomé Magalhães has defended her Ph D thesis in Biotechnology at Instituto Superior Técnico, monday the 27th January 2015. During the last years, and under the supervision of Gabriel Monteiro from BERG-IBB and Inge Nielsen from Nofima, Salomé has investigated strategies  to increase the resistance of DNA vaccines against nuclease, which include the use of  inhibitors from marine origin and the modification of specific nucleotides in the vaccines backbone.  The title of her thesis is “Strategies to Increase DNA Vaccines Resistance to Nucleases”. 

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Determination of Recombination Efficiency in Minicircle Production by Real-time PCR

Determination of Recombination Efficiency in Minicircle Production by Real-time PCR | iBB | Scoop.it

The determination of the recombination efficiency and the monitoring of parental plasmids (PP), minicircles (MC) and miniplasmid (MP) species during processing and in the final product are critical aspects of minicircle manufacturing. A paper published in Analytical Biochemistry by BERG-iBB researchers Cláudia Alves, Miguel Prazeres and Gabriel Monteiro describes a real-time PCR method for the specific identification of PP, MP or MC that uses sets of primers specific for each species. The results presented indicate that the method can be applied for the determination of recombination efficiency in pure DNA samples.

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Minicircle‐based Expression of VEGF in Mesenchymal Stromal Cells from Human Tissues

Minicircle‐based Expression of VEGF in Mesenchymal Stromal Cells from Human Tissues | iBB | Scoop.it

Mesenchymal stromal cells (MSC) have been exploited for the treatment of ischemic diseases given their angiogenic potential. A recent study published in the Journal of Gene Medicine by BERG and SCERG researchers compares the angiogenic potential of MSC obtained from bone marrow (BM), adipose tissue (AT) and umbilical cord matrix (UCM) that were genetically modified with VEGF‐encoding minicircle vectors. Transfected cells displayed higher in vitro angiogenic potential than non‐transfected controls, as demonstrated by functional in vitro assays, but no significant differences were observed among cells from different sources.

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Minicircle Biopharmaceuticals: An Overview of Purification Strategies

Minicircle Biopharmaceuticals: An Overview of Purification Strategies | iBB | Scoop.it

Minicircles are non-viral delivery vectors with promising features for biopharmaceutical applications. These vectors are obtained in vivo in Escherichia coli by the intramolecular recombination of a parental plasmid, which generates the minicircle of interest and an unwanted miniplasmid. Several strategies have been proposed over the years to meet the challenge of purifying and obtaining high quality minicircles. The characteristics of the strain and parental plasmid used have a high impact and strongly affect the purification strategy that can be applied. In a review published in Frontiers in Chemical Engineering, Cláudia Alves, Miguel Prazeres and Gabriel Monteiro from BERG-IST summarize the different methods developed so far, focusing not only on the purification method itself but also on its dependence on the upstream production strategy used.

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Featured Photo: CRISPR/Cas9 Engineering of E. coli for Minicircle Production

Featured Photo: CRISPR/Cas9 Engineering of E. coli for Minicircle Production | iBB | Scoop.it

Description: plate for screening CRISPR modifications, featured photo by Cláudia Alves, Copyright BERG-iBB and University of Florida 2019.

 

Context: BIOTecnico PhD student Cláudia Alves, under the supervision of BERG-iBB researchers Gabriel Monteiro and Miguel Prazeres and in collaboration with Christopher Reisch from the University of Florida, is using CRISPR/Cas9 to create mutations in E. coli that improve minicircle production.

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Increasing Plasmid Copy Number in Lactococcus lactis

Increasing Plasmid Copy Number in Lactococcus lactis | iBB | Scoop.it

The lipopolysaccharide-free Lactococcus lactis is an interesting alternative to E. coli for plasmid production. A key requirement for L. lactis‐based plasmid manufacturing is the availability of high‐copy number plasmids. In a recent publication in Biotechnology Journal, BERG-iBB researchers led by Gabriel Monteiro describe how engineering of the repDE Ribosome Binding Site led to an increase in the copy number  of plasmid pTRKH3 in L. lactis LMG19460 cells.  The number of copies of mutant pTRKH3‐b increased up to 215 copies per chromosome, which corresponds to a 3.5 fold increase when compared to the non‐modified pTRKH3. The new mutant is an important step towards the establishment of lactic acid bacteria as viable plasmid producers. The work was developed in the context of the FCT-funded, LactoSynt project.

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Multimodal Chromatography of Supercoiled Minicircles

Multimodal Chromatography of Supercoiled Minicircles | iBB | Scoop.it

The ability to purify supercoiled minicircles (sc MC) from related miniplasmid (MP) and parental plasmid (PP) impurities is critical for a wider application of MC vectors in gene therapy research. BERG-iBB researchers developed a process that relies on multimodal chromatography with a 2-step NaCl elution to separate sc MC from open circular impurities. A detailed study of the DNA-ligand interactions underlying the separation shows that the order of elution reflects the increasing degree of base exposure of the species. Data further suggests that nucleic acid bases most likely engage in cation-π, π-π stacking and H-bonding with ligand. The work was published in Separation and Purification Technology.

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Production and Purification of Supercoiled Minicircles

Production and Purification of Supercoiled Minicircles | iBB | Scoop.it

A wider application of minicircle (MC) vectors in gene therapy research depends critically on the ability to purify supercoiled MC from related miniplasmid (MP) and parental plasmid (PP) impurities. BERG-iBB researchers published a protocol that describes a purification strategy that combines the in vitro enzymatic relaxation of supercoiled MP and PP impurities by a nicking endonuclease, and topoisomer separation and RNA clearance by hydrophobic interaction chromatography. The time required to follow the full protocol, from production to isolation of sc MC is, approximately, 50 hr. The process delivers supercoiled MCs that are virtually free from MP, PP, RNA and protein impurities. The work was published in Human Gene Therapy Methods.

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Lactic Acid Bacterial Factories

Lactic Acid Bacterial Factories | iBB | Scoop.it

The project "LactoSynt: Lactic Acid Bacteria as Cell Factories: a Synthetic Biology Approach for Plasmid DNA and Recombinant Protein Production" has been recommended for funding by FCT (2017 Call for SR&TD Project Grants). The goal of LactoSynt is to engineer lactic acid bacteria and plasmid vectors in order to develop a flexible platform for biomolecule production. Applications in the pharmaceutical field (DNA vaccines, recombinant proteins) are envisaged. The project, which falls within the scientific area of Medical Biotechnology, is headed by Gabriel Monteiro from BERG-iBB and Leonilde Moreira from BSRG-iBB.

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Sofia Duarte Defends PhD Thesis in Biotechnology and Biosciences

Sofia Duarte Defends PhD Thesis in Biotechnology and Biosciences | iBB | Scoop.it

Sofia de Oliveira Duarte has defended her Ph D thesis in Biotechnology and Biosciences at Instituto Superior Técnico on the 16th April 2018. During the last years, and under the supervision of Gabriel Monteiro from BERG-IBB and Lígia Rodrigues from University of Minho, Sofia has investigated the possibility of engineering lactic acid bacteria to produce plasmids and proteins. The title of her thesis is "Lactic acid bacteria as cell factories: A synthetic biology approach for plasmid DNA and recombinant protein production”.

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Towards Effective Non-viral Gene Delivery Vectors

Towards Effective Non-viral Gene Delivery Vectors | iBB | Scoop.it

Despite very good safety records, clinical trials using plasmid DNA have failed due to low transfection efficiency and brief transgene expression. One of the reasons for this failure is poor plasmid design. In a paper published in Biotechnology and Genetic Engineering Reviews, researchers from iBB-BERG led by Gabriel Monteiro review critical aspects of plasmid design including promoter, polyadenylation signal, codon optimization and/or insertion of introns or nuclear-targeting sequences. The impact of detrimental elements like CpG motifs, selection markers, origins of replication, cryptic eukaryotic signals or nuclease-susceptible regions and inverted repeats is also examined. Click on title to learn more.


Photo details: CHO cells transfected with plasmid vector coding for GFP.

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Improvement of DNA Minicircle Production

Improvement of DNA Minicircle Production | iBB | Scoop.it
The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, a team led by Gabriel Monteiro and Miguel Prazeres from iBB-BERG and Kristala Prather from MIT, optimized the 5′ untranslated region (5′-UTR) of parA messenger RNA (mRNA) using a predictive thermodynamic model. The optimized parA gene was then inserted in the chromosome of an E. coli strain with improved arabinose uptake. overall the results show that the improvements made in the 5′-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation.The research was developed under the framework of the MIT-Portugal program and is a direct result of Michaela Simcikova's Ph D. thesis in Bioengineering. Click on title to read the publication in Applied Microbiology and Biotechnology.
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A Review on Plasmid Biopharmaceuticals

A Review on Plasmid Biopharmaceuticals | iBB | Scoop.it

Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its  mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. In a chapter published in the textbook Plasmids: Biology and Impact in Biotechnology and Discovery, edited by the American Society for Microbiology, BERG researchers Miguel Prazeres and Gabriel Monteiro provide a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The textbook is intended for upper-level undergraduate and graduate courses in the biosciences.

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