Neonatal screening (NBS) was initiated in Europe during the 1960s with the screening for phenylke... more Neonatal screening (NBS) was initiated in Europe during the 1960s with the screening for phenylketonuria. The panel of screened disorders (“conditions”) then gradually expanded, with a boost in the late 1990s with the introduction of tandem mass spectrometry (MS/MS), making it possible to screen for 40–50 conditions using a single blood spot. The most recent additions to screening programmes (screening for cystic fibrosis, severe combined immunodeficiency and spinal muscular atrophy) were assisted by or realised through the introduction of molecular technologies. For this survey, we collected data from 51 European countries. We report the developments between 2010 and 2020 and highlight the achievements reached with the progress made in this period. We also identify areas where further progress can be made, mainly by exchanging knowledge and learning from experiences in neighbouring countries. Between 2010 and 2020, most NBS programmes in geographical Europe matured considerably, bo...
In the present study, an aqueous two-phase partitioning system (ATPS) was developed and evaluated... more In the present study, an aqueous two-phase partitioning system (ATPS) was developed and evaluated as an initial fractionation step for therapeutic antibodies and enzymes from tobacco extracts. A detailed study has been performed to analyze the effect of pH, ionic composition of the system, types of polymers and their molecular weight and concentration, on the partitioning behavior of tobacco proteins and human anti-human immunodeficiency virus (HIV) monoclonal antibody 2F5 (mAb 2F5). A polyethyleneglycol/phosphate (PEG/Pi) aqueous two-phase system composed of 12% (w/w) PEG 1500 and 13% (w/w) phosphate buffer, pH 5, was selected as the system with the highest selectivity of antibody over native tobacco proteins. Under selected conditions, sufficient purification (3-4-fold) with high recovery at the bottom phase (approximately 95%) was achieved for mAb 2F5. In addition, the system allows removal of plant-derived compounds, such as phenolics and toxic alkaloids. The antibody fraction may be directly applied to a Protein A affinity column without any further pre-treatment, thus allowing homogenous antibody preparation. Analysis of the purified antibody fraction by enzyme-linked immunosorbent assay (ELISA) and western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was further demonstrated using additional proteins and enzymes of therapeutic importance, such as neuraminidase (NA) from influenza virus and human anti-HIV monoclonal antibody 2G12 (mAb 2G12), and showed that the system may find wide applicability as an economic extraction strategy for the initial fractionation of biopharmaceuticals from transgenic tobacco plants.
The lock-and-key (LAK) motif, a common structural moiety found in subunit interfaces of glutathio... more The lock-and-key (LAK) motif, a common structural moiety found in subunit interfaces of glutathione S-transferases (GSTs), plays an important role in biomolecular recognition and quaternary structure integrity. Inspection of the key structural features of the LAK motif prompted the de novo design and combinatorial synthesis of a 13-membered solid-phase ligand library, employing as a lead ligand the Phe-Trz-X structure, mimicking the LAK motif. 1,3,5-Triazine (Trz) was used as the scaffold for assembly, substituted with different LAK-mimetic amino acids. De novo ligand design was effected using bioinformatics and molecular modeling and based on mimicking the interactions of the LAK motif. The library of affinity adsorbents was assessed for binding corn and human serum proteomes and purified proteins of different structure and ligand binding specificity. The results showed remarkable differences in the binding specificity of LAK-mimetic adsorbents for a wide range of proteins, as a consequence of minor changes in ligand structure. One LAK-mimetic adsorbent was integrated in a single-step purification protocol for human monoclonal anti-human immunodeficiency virus 2F5 antibody (mAb 2F5) from spiked corn extract, affording high recovery and purity. The results demonstrate that the principle of natural recognition found in the lock-and-key motif, in combination with de novo combinatorial design, may lead to synthetic affinity ligands, useful in downstream processing and proteomic research.
Interferons (IFNs) are a family of pleiotropic cytokines used for the treatment of various viral ... more Interferons (IFNs) are a family of pleiotropic cytokines used for the treatment of various viral infections and cancers. The low-cost production of IFNs with high biological value and the discovery of IFNs with improved properties are important for the treatment of these diseases as well as for understanding the physiological functions of these compounds. We describe a protein expression system for the production of IFNs alpha2, alpha8, and their hybrids in insoluble form in Escherichia coli, coupled to an efficient two-step optimized refolding and histidine-tag purification protocol. The expressed IFNs were of high biological value, as shown in antiviral and antiproliferative assays and some had specific activities higher than those of the commercially available interferon preparations and exhibited novel properties. This time-efficient, optimized protein expression method allows for the production of not just a single interferon subtype but several native and hybrid IFNs with relatively high yield and low cost that can be used in functional and potentially clinical assays.
Journal of Interferon & Cytokine Research, Jan 1, 2003
Type I interferons (IFNs) are a family of pleiotropic cytokines with antiviral, antiproliferative... more Type I interferons (IFNs) are a family of pleiotropic cytokines with antiviral, antiproliferative, and immunomodulatory properties. The type I IFN family consists of 12 IFN-alpha subtypes, IFN-beta, and IFN-omega. Cells lacking the receptor-associated protein kinase Tyk2 (U1A) are responsive only to IFN-beta and partially to IFN-alpha8. We constructed a series of IFN-alpha2/alpha8 hybrids and mutants and identified the region within IFN-alpha8 responsible for its activity in Tyk2-deficient cells. The same domain mediates the interactions between IFN and IFN-alpha receptor (IFNAR) in Tyk2-complemented and Tyk2-deficient cells (U1A). The presence or absence of Tyk2 altered the inhibitory effects of anti-IFNAR antibodies, suggesting that the IFN-alpha binding domain on IFNAR is altered by the presence of Tyk2. The activity of IFN-beta was not significantly affected by the deletion of Tyk2, and, surprisingly, one of our IFN-alpha2/alpha8 hybrids (IFN-alpha288) behaved like IFN-beta in a number of assays that distinguish IFN-alphas from IFN-beta. This suggests that this hybrid mimics the interactions of IFN-beta with the receptor and also suggests the existence of a distinct binding site(s) on IFNAR for IFN-beta and some hybrid IFN-alphas.
Influenza NA (neuraminidase) is an antiviral target of high pharmaceutical interest because of it... more Influenza NA (neuraminidase) is an antiviral target of high pharmaceutical interest because of its essential role in cleaving sialic acid residues from cell surface glycoproteins and facilitating release of virions from infected cells. The present paper describes the use of structural information in the progressive design from a lead binding ion (a sulfate) to a potent submicromolor inhibitor (Ki 0.13 μM). Structural information derived from the X-ray structure of an NA complexed with several sulfate ions, in combination with results derived from affinity labelling and molecular modelling studies, was used to guide design of potent sulfonic acid-based inhibitors. These inhibitors are structural fragments of the polysulfonate triazine dye Cibacron Blue 3GA and represent novel lead scaffolds for designing non-carbohydrate inhibitors for influenza neuraminidases.
Over the last decade there has been significant progress in understanding the molecular basis of ... more Over the last decade there has been significant progress in understanding the molecular basis of disease processes. At the same time the technological advances in the area of genomics and the efforts in proteomics research have increased the possibility of discovering many proteins with defined therapeutic functions. A large number of these proteins have found clinical application. Despite the importance of proteins as therapeutic agents, they have a number of disadvantages in comparison to small-molecule drugs, including immunogenicity and antigenicity, poor efficacy and oral bioavailability as well as, in many cases, short serum half-lives. To date, the most promising approaches for improving protein therapeutics rely on the use of genetic engineering and site-specific chemical synthesis/modification techniques. Improving the potency of protein drugs by employing modern recombinant DNA technologies and novel chemical synthesis techniques is of primary importance, not only because of the enormous medicinal benefit but also because of the significant economic edge an improved drug can provide in today's competitive market.
Type I interferons (IFNs) are a family of pleiotropic cytokines with antiviral, antiproliferative... more Type I interferons (IFNs) are a family of pleiotropic cytokines with antiviral, antiproliferative, and immunomodulatory properties. The type I IFN family consists of 12 IFN-alpha subtypes, IFN-beta, and IFN-omega. Cells lacking the receptor-associated protein kinase Tyk2 (U1A) are responsive only to IFN-beta and partially to IFN-alpha8. We constructed a series of IFN-alpha2/alpha8 hybrids and mutants and identified the region within IFN-alpha8 responsible for its activity in Tyk2-deficient cells. The same domain mediates the interactions between IFN and IFN-alpha receptor (IFNAR) in Tyk2-complemented and Tyk2-deficient cells (U1A). The presence or absence of Tyk2 altered the inhibitory effects of anti-IFNAR antibodies, suggesting that the IFN-alpha binding domain on IFNAR is altered by the presence of Tyk2. The activity of IFN-beta was not significantly affected by the deletion of Tyk2, and, surprisingly, one of our IFN-alpha2/alpha8 hybrids (IFN-alpha288) behaved like IFN-beta in a number of assays that distinguish IFN-alphas from IFN-beta. This suggests that this hybrid mimics the interactions of IFN-beta with the receptor and also suggests the existence of a distinct binding site(s) on IFNAR for IFN-beta and some hybrid IFN-alphas.
Monoclonal anti-HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies a... more Monoclonal anti-HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L-Phe/beta-Ala bi-substituted 1,3,5-triazine (Trz) scaffold (beta-Ala-Trz-L-Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10-binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K(D)) of 0.41 +/- 0.05 microM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60-80%). Analysis of the antibody preparation by SDS-PAGE, enzyme-linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids.
The interferons: characterization and …, Jan 1, 2006
73 3 Interferon Proteins: Structure, Production and Purification Dimitris Platis and Graham R. Fos... more 73 3 Interferon Proteins: Structure, Production and Purification Dimitris Platis and Graham R. Foster 3.1 Introduction The type I interferons (IFNs) are a family of closely related cytokines consisting of 12 different IFN-a subtypes, one IFN-b subtype and one IFN-o subtype [1, 2]. Of the ...
Publication View. 32403717. Structure/function analysis of Type I interferons / (2003). Platis, D... more Publication View. 32403717. Structure/function analysis of Type I interferons / (2003). Platis, Dimitris. Abstract. Thesis (Ph.D.)--University of London, 2003. Publication details. Download, http://worldcat.org/oclc/59174740. Publisher, London : University of London,. ...
The type I interferons (IFNs) are a family of closely related cytokines consisting of 12 differen... more The type I interferons (IFNs) are a family of closely related cytokines consisting of 12 different IFN-α subtypes, one IFN-/ subtype and one IFN-o subtype [1, 2]. Of the many different type I IFNs that are produced naturally, only IFN-α2 and -/ are currently in widespread clinical use. In ...
Neonatal screening (NBS) was initiated in Europe during the 1960s with the screening for phenylke... more Neonatal screening (NBS) was initiated in Europe during the 1960s with the screening for phenylketonuria. The panel of screened disorders (“conditions”) then gradually expanded, with a boost in the late 1990s with the introduction of tandem mass spectrometry (MS/MS), making it possible to screen for 40–50 conditions using a single blood spot. The most recent additions to screening programmes (screening for cystic fibrosis, severe combined immunodeficiency and spinal muscular atrophy) were assisted by or realised through the introduction of molecular technologies. For this survey, we collected data from 51 European countries. We report the developments between 2010 and 2020 and highlight the achievements reached with the progress made in this period. We also identify areas where further progress can be made, mainly by exchanging knowledge and learning from experiences in neighbouring countries. Between 2010 and 2020, most NBS programmes in geographical Europe matured considerably, bo...
In the present study, an aqueous two-phase partitioning system (ATPS) was developed and evaluated... more In the present study, an aqueous two-phase partitioning system (ATPS) was developed and evaluated as an initial fractionation step for therapeutic antibodies and enzymes from tobacco extracts. A detailed study has been performed to analyze the effect of pH, ionic composition of the system, types of polymers and their molecular weight and concentration, on the partitioning behavior of tobacco proteins and human anti-human immunodeficiency virus (HIV) monoclonal antibody 2F5 (mAb 2F5). A polyethyleneglycol/phosphate (PEG/Pi) aqueous two-phase system composed of 12% (w/w) PEG 1500 and 13% (w/w) phosphate buffer, pH 5, was selected as the system with the highest selectivity of antibody over native tobacco proteins. Under selected conditions, sufficient purification (3-4-fold) with high recovery at the bottom phase (approximately 95%) was achieved for mAb 2F5. In addition, the system allows removal of plant-derived compounds, such as phenolics and toxic alkaloids. The antibody fraction may be directly applied to a Protein A affinity column without any further pre-treatment, thus allowing homogenous antibody preparation. Analysis of the purified antibody fraction by enzyme-linked immunosorbent assay (ELISA) and western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was further demonstrated using additional proteins and enzymes of therapeutic importance, such as neuraminidase (NA) from influenza virus and human anti-HIV monoclonal antibody 2G12 (mAb 2G12), and showed that the system may find wide applicability as an economic extraction strategy for the initial fractionation of biopharmaceuticals from transgenic tobacco plants.
The lock-and-key (LAK) motif, a common structural moiety found in subunit interfaces of glutathio... more The lock-and-key (LAK) motif, a common structural moiety found in subunit interfaces of glutathione S-transferases (GSTs), plays an important role in biomolecular recognition and quaternary structure integrity. Inspection of the key structural features of the LAK motif prompted the de novo design and combinatorial synthesis of a 13-membered solid-phase ligand library, employing as a lead ligand the Phe-Trz-X structure, mimicking the LAK motif. 1,3,5-Triazine (Trz) was used as the scaffold for assembly, substituted with different LAK-mimetic amino acids. De novo ligand design was effected using bioinformatics and molecular modeling and based on mimicking the interactions of the LAK motif. The library of affinity adsorbents was assessed for binding corn and human serum proteomes and purified proteins of different structure and ligand binding specificity. The results showed remarkable differences in the binding specificity of LAK-mimetic adsorbents for a wide range of proteins, as a consequence of minor changes in ligand structure. One LAK-mimetic adsorbent was integrated in a single-step purification protocol for human monoclonal anti-human immunodeficiency virus 2F5 antibody (mAb 2F5) from spiked corn extract, affording high recovery and purity. The results demonstrate that the principle of natural recognition found in the lock-and-key motif, in combination with de novo combinatorial design, may lead to synthetic affinity ligands, useful in downstream processing and proteomic research.
Interferons (IFNs) are a family of pleiotropic cytokines used for the treatment of various viral ... more Interferons (IFNs) are a family of pleiotropic cytokines used for the treatment of various viral infections and cancers. The low-cost production of IFNs with high biological value and the discovery of IFNs with improved properties are important for the treatment of these diseases as well as for understanding the physiological functions of these compounds. We describe a protein expression system for the production of IFNs alpha2, alpha8, and their hybrids in insoluble form in Escherichia coli, coupled to an efficient two-step optimized refolding and histidine-tag purification protocol. The expressed IFNs were of high biological value, as shown in antiviral and antiproliferative assays and some had specific activities higher than those of the commercially available interferon preparations and exhibited novel properties. This time-efficient, optimized protein expression method allows for the production of not just a single interferon subtype but several native and hybrid IFNs with relatively high yield and low cost that can be used in functional and potentially clinical assays.
Journal of Interferon & Cytokine Research, Jan 1, 2003
Type I interferons (IFNs) are a family of pleiotropic cytokines with antiviral, antiproliferative... more Type I interferons (IFNs) are a family of pleiotropic cytokines with antiviral, antiproliferative, and immunomodulatory properties. The type I IFN family consists of 12 IFN-alpha subtypes, IFN-beta, and IFN-omega. Cells lacking the receptor-associated protein kinase Tyk2 (U1A) are responsive only to IFN-beta and partially to IFN-alpha8. We constructed a series of IFN-alpha2/alpha8 hybrids and mutants and identified the region within IFN-alpha8 responsible for its activity in Tyk2-deficient cells. The same domain mediates the interactions between IFN and IFN-alpha receptor (IFNAR) in Tyk2-complemented and Tyk2-deficient cells (U1A). The presence or absence of Tyk2 altered the inhibitory effects of anti-IFNAR antibodies, suggesting that the IFN-alpha binding domain on IFNAR is altered by the presence of Tyk2. The activity of IFN-beta was not significantly affected by the deletion of Tyk2, and, surprisingly, one of our IFN-alpha2/alpha8 hybrids (IFN-alpha288) behaved like IFN-beta in a number of assays that distinguish IFN-alphas from IFN-beta. This suggests that this hybrid mimics the interactions of IFN-beta with the receptor and also suggests the existence of a distinct binding site(s) on IFNAR for IFN-beta and some hybrid IFN-alphas.
Influenza NA (neuraminidase) is an antiviral target of high pharmaceutical interest because of it... more Influenza NA (neuraminidase) is an antiviral target of high pharmaceutical interest because of its essential role in cleaving sialic acid residues from cell surface glycoproteins and facilitating release of virions from infected cells. The present paper describes the use of structural information in the progressive design from a lead binding ion (a sulfate) to a potent submicromolor inhibitor (Ki 0.13 μM). Structural information derived from the X-ray structure of an NA complexed with several sulfate ions, in combination with results derived from affinity labelling and molecular modelling studies, was used to guide design of potent sulfonic acid-based inhibitors. These inhibitors are structural fragments of the polysulfonate triazine dye Cibacron Blue 3GA and represent novel lead scaffolds for designing non-carbohydrate inhibitors for influenza neuraminidases.
Over the last decade there has been significant progress in understanding the molecular basis of ... more Over the last decade there has been significant progress in understanding the molecular basis of disease processes. At the same time the technological advances in the area of genomics and the efforts in proteomics research have increased the possibility of discovering many proteins with defined therapeutic functions. A large number of these proteins have found clinical application. Despite the importance of proteins as therapeutic agents, they have a number of disadvantages in comparison to small-molecule drugs, including immunogenicity and antigenicity, poor efficacy and oral bioavailability as well as, in many cases, short serum half-lives. To date, the most promising approaches for improving protein therapeutics rely on the use of genetic engineering and site-specific chemical synthesis/modification techniques. Improving the potency of protein drugs by employing modern recombinant DNA technologies and novel chemical synthesis techniques is of primary importance, not only because of the enormous medicinal benefit but also because of the significant economic edge an improved drug can provide in today's competitive market.
Type I interferons (IFNs) are a family of pleiotropic cytokines with antiviral, antiproliferative... more Type I interferons (IFNs) are a family of pleiotropic cytokines with antiviral, antiproliferative, and immunomodulatory properties. The type I IFN family consists of 12 IFN-alpha subtypes, IFN-beta, and IFN-omega. Cells lacking the receptor-associated protein kinase Tyk2 (U1A) are responsive only to IFN-beta and partially to IFN-alpha8. We constructed a series of IFN-alpha2/alpha8 hybrids and mutants and identified the region within IFN-alpha8 responsible for its activity in Tyk2-deficient cells. The same domain mediates the interactions between IFN and IFN-alpha receptor (IFNAR) in Tyk2-complemented and Tyk2-deficient cells (U1A). The presence or absence of Tyk2 altered the inhibitory effects of anti-IFNAR antibodies, suggesting that the IFN-alpha binding domain on IFNAR is altered by the presence of Tyk2. The activity of IFN-beta was not significantly affected by the deletion of Tyk2, and, surprisingly, one of our IFN-alpha2/alpha8 hybrids (IFN-alpha288) behaved like IFN-beta in a number of assays that distinguish IFN-alphas from IFN-beta. This suggests that this hybrid mimics the interactions of IFN-beta with the receptor and also suggests the existence of a distinct binding site(s) on IFNAR for IFN-beta and some hybrid IFN-alphas.
Monoclonal anti-HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies a... more Monoclonal anti-HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L-Phe/beta-Ala bi-substituted 1,3,5-triazine (Trz) scaffold (beta-Ala-Trz-L-Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10-binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K(D)) of 0.41 +/- 0.05 microM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60-80%). Analysis of the antibody preparation by SDS-PAGE, enzyme-linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids.
The interferons: characterization and …, Jan 1, 2006
73 3 Interferon Proteins: Structure, Production and Purification Dimitris Platis and Graham R. Fos... more 73 3 Interferon Proteins: Structure, Production and Purification Dimitris Platis and Graham R. Foster 3.1 Introduction The type I interferons (IFNs) are a family of closely related cytokines consisting of 12 different IFN-a subtypes, one IFN-b subtype and one IFN-o subtype [1, 2]. Of the ...
Publication View. 32403717. Structure/function analysis of Type I interferons / (2003). Platis, D... more Publication View. 32403717. Structure/function analysis of Type I interferons / (2003). Platis, Dimitris. Abstract. Thesis (Ph.D.)--University of London, 2003. Publication details. Download, http://worldcat.org/oclc/59174740. Publisher, London : University of London,. ...
The type I interferons (IFNs) are a family of closely related cytokines consisting of 12 differen... more The type I interferons (IFNs) are a family of closely related cytokines consisting of 12 different IFN-α subtypes, one IFN-/ subtype and one IFN-o subtype [1, 2]. Of the many different type I IFNs that are produced naturally, only IFN-α2 and -/ are currently in widespread clinical use. In ...
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