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| 3. |
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| 4. |
- Azizi, Tamir, et al.
(författare)
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A COST Action on microbial responses to low pH : Developing links and sharing resources across the academic-industrial divide
- 2022
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 72, s. 64-70
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Tidskriftsartikel (refereegranskat)abstract
- We present work of our COST Action on "Understanding and exploiting the impacts of low pH on micro-or-ganisms". First, we summarise a workshop held at the European Federation of Biotechnology meeting on Mi-crobial Stress Responses (online in 2020) on "Industrial applications of low pH stress on microbial bio-based production", as an example of an initiative fostering links between pure and applied research. We report the outcomes of a small survey on the challenging topic of developing links between researchers working in academia and industry that show that, while people in different sectors strongly support such links, barriers remain that obstruct this process. We present the thoughts of an expert panel held as part of the workshop above, where people with experience of collaboration between academia and industry shared ideas on how to develop and maintain links. Access to relevant information is essential for research in all sectors, and because of this we have developed, as part of our COST Action goals, two resources for the free use of all researchers with interests in any aspects of microbial responses to low pH. These are (1) a comprehensive database of references in the literature on different aspects of acid stress responses in different bacterial and fungal species, and (2) a database of research expertise across our network. We invite the community of researchers working in this field to take advantage of these resources to identify relevant literature and opportunities for establishing collaborations.
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| 10. |
- Carbonaro, Miriam, et al.
(författare)
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Genomic mining of Geobacillus stearothermophilus GF16 for xylose production from hemicellulose-rich biomasses using secreted enzymes
- 2024
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Ingår i: New Biotechnology. - 1876-4347 .- 1871-6784. ; 82, s. 14-24
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Tidskriftsartikel (refereegranskat)abstract
- The valorization of lignocellulosic biomass, derived from various bio-waste materials, has received considerable attention as a sustainable approach to improve production chains while reducing environmental impact. Microbial enzymes have emerged as key players in the degradation of polysaccharides, offering versatile applications in biotechnology and industry. Among these enzymes, glycoside hydrolases (GHs) play a central role. Xylanases, in particular, are used in a wide range of applications and are essential for the production of xylose, which can be fermented into bioethanol or find use in many other industries. Currently, fungal secretomes dominate as the main reservoir of lignocellulolytic enzymes, but thermophilic microorganisms offer notable advantages in terms of enzyme stability and production efficiency. Here we present the genomic characterization of Geobacillus stearothermophilus GF16 to identify genes encoding putative enzymes involved in lignocellulose degradation. Thermostable GHs secreted by G. stearothermophilus GF16 were investigated and found to be active on different natural polysaccharides and synthetic substrates, revealing an array of inducible GH activities. In particular, the concentrated secretome possesses significant thermostable xylanase and β-xylosidase activities (5 ×103 U/L and 1.7 ×105 U/L, respectively), highlighting its potential for application in biomass valorization. We assessed the hemicellulose hydrolysis capabilities of various agri-food wastes using the concentrated secretome of the strain cultivated on xylan. An impressive 300-fold increase in xylose release compared to a commercially available cocktail was obtained with the secretome, underscoring the remarkable efficacy of this approach.
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| 11. |
- Carlsson, Fredrika, et al.
(författare)
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Novel antibody specificities targeting glycoprotein B of cytomegalovirus identified by molecular library technology
- 2009
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 25:6, s. 429-436
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies provide some protection against cytomegalovirus-mediated disease. All aspects of antibody recognition of important viral antigens are however not fully appreciated. Glycoprotein B (gB), a key protein in the viral membrane, participates in viral infection and it is a component of prototype vaccines. By using combinatorial antibody library and selection technology, novel antibody specificities targeting gB have now been isolated. We define a monoclonal antibody fragment able to recognize site I of antigenic domain (AD) 2, a poorly immunogenic epitope targeted by potent virus-neutralizing antibodies, in a way that is different from the binding of antibodies induced by infection but similar to those induced by vaccination. We also describe the existence of a novel epitope overlapping site I of AD-2 and AD-1, the immunodominant epitope of gB. These specificities, derived by molecular engineering, will be useful for the future assessment of humoral immune responses against this opportunistic viral infection.
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| 12. |
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| 13. |
- Cerullo, Gabriella, et al.
(författare)
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Directed evolution of the type C feruloyl esterase from Fusarium oxysporum FoFaeC and molecular docking analysis of its improved variants
- 2019
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Ingår i: New Biotechnology. - : Elsevier. - 1871-6784 .- 1876-4347. ; 51, s. 14-20
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Tidskriftsartikel (refereegranskat)abstract
- The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica. Screening for enzymatic activity towards the substrates 5-bromo-4-chloroindol-3-yl and 4-nitrocatechol-1-yl ferulates allowed the selection of 96 enzyme variants endowed with improved enzymatic activity that were then characterized for thermo- and solvent- tolerance. The five best mutants in terms of higher activity, thermo- and solvent- tolerance were selected for analysis of substrate specificity. Variant L432I was shown to be able to hydrolyze all the tested substrates, except methyl sinapate, with higher activity than wild type FoFaeC towards methyl p-coumarate, methyl ferulate and methyl caffeate. Moreover, the E455D variant was found to maintain completely its hydrolytic activity after two hour incubation at 55 °C, whereas the L284Q/V405I variant showed both higher thermo- and solvent- tolerance than wild type FoFaeC. Small molecule docking simulations were applied to the five novel selected variants in order to examine the binding pattern of substrates used for enzyme characterization of wild type FoFaeC and the evolved variants.
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| 14. |
- Christakopoulos, Paul, et al.
(författare)
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Synthesis of biological active compounds using carbohydrate esterases as biocatalysts
- 2014
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 31:Supplement, s. S90-S91
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Tidskriftsartikel (refereegranskat)abstract
- Various fungal and bacterial carbohydrate esterases represent appealing biocatalysts that have the ability not only to deconstruct plant biomass but also to modify compounds with a potential use in food, cosmetic and pharmaceutical industries. Feruloyl esterases (FAEs, E.C. 3.1.1.73) have been proved promising candidates for the enzymatic synthesis of antioxidants allowing more flexible process configurations. Among the advantages they provide are use of lower temperatures (50-60 °C) comparing to the counterpart chemical process (150οC), one step production of one product instead of mixtures and no need of by-product and catalyst residues removal in order to produce clean and high quality substances. Glucuronoyl esterase (GE) synthetic ability needs to be explored towards the production of alkyl branched glucuronic acid derivatives which are non-ionic surfactants and have good surface properties, including biodegradability. In addition, due to their tastelessness, non skin-irritation and non toxicity, these bioactive compounds find diverse uses in the cosmetic and pharmaceutical industries.Aim of this work is the development of competitive and eco-friendly bioconversions based on transesterification reactions catalyzed by FAEs and GEs, for the production of molecules with antioxidant activity, such as phenolic fatty and sugar esters. The synthesis of four biological active compounds (prenyl ferulate, prenyl caffeate, 5-O-(trans-feruloyl)-arabinofuranose, and glyceryl ferulate) was evaluated using recombinant FAEs from Myceliopthora thermophila and Fusarium oxysporum, while the synthesis of benzyl D-glucuronate and prenyl-D-glucuronate was evaluated using recombinant GEs from M. thermophila. All reactions were carried out in ternary systems of n-hexane/alcohol/water forming surfactantless microemulsions.
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| 15. |
- de Oliveira, Felipe Marques Souza, et al.
(författare)
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Detection of post-translational modifications using solid-phase proximity ligation assay.
- 2018
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Ingår i: New biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 45:October, s. 51-59
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Tidskriftsartikel (refereegranskat)abstract
- Post-translational modifications (PTMs) regulate protein activities to help orchestrate and fine-tune cellular processes. Dysregulation of PTMs is often related with disorders and malignancies, and may serve as a precise biomarker of disease. Developing sensitive tools to measure and monitor low-abundant PTMs in tissue lysates or serum will be instrumental for opening up new PTM-based diagnostic avenues. Here, we investigate the use of solid-phase proximity ligation assay (SP-PLA) for detection of different PTMs. The assay depends on the recognition of the target protein molecule and its modification by three affinity binders. Using antibodies and lectins, we applied the method for detection of glycosylated CD44 and E-Cadherin, and phosphorylated p53 and EGFR. The assay was found to have superior dynamic range and limit of detection compared to standard ELISAs. In summary, we have established the use of SP-PLA as an appropriate method for sensitive detection of PTMs in lysates and sera, which may provide a basis for future PTM-based diagnostic and prognostic biomarkers.
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| 16. |
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| 18. |
- Ewing, Andrew, 1957
(författare)
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Mass spectrometry imaging of lipids and metabolites: from fruit fly brains to single nanometer vesicles
- 2016
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 33:Supplement: S Meeting Abstract: O5-1, s. S21-S21
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Tidskriftsartikel (refereegranskat)abstract
- We have been developing mass spectrometry imaging methods to study the process of neurocommunication at the system and cellular level. We focus on PC12 cells as a model of exocytosis and the fly model (Drosophila melanogaster) providing a unique system to examine neurotransmitter release and drug dependence mechanisms in a small, but complete system.Mass spectrometry imaging with ion beams allows spatial resolution of a few micrometers down to 40 nanometers in favorable cases. We have been using secondary ion mass spectrometry (SIMS) with a unique 40-kV argon cluster ion source and the NanoSIMS to measure the lipids across the fly brain and catecholamine in nanometer vesicles, respectively. Here, we have focused on the effect of the drug, methylphenidate, on lipid composition in the brain and find that it varies in a way that might affect learning and memory. We have also used NanoSIMS to measure transmitter in subregions of nanometer vesicles. Combined with other new methods to measure the content of the interior of vesicles, we have begun to investigate the details and implications of open and closed exocytosis on regulation of how the brain works.
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| 19. |
- Flanigon, James, et al.
(författare)
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Multiplex protein detection with DNA readout via mass spectrometry
- 2013
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 30:2, s. 153-158
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Tidskriftsartikel (refereegranskat)abstract
- Multiplex protein quantification has been constrained by issues of assay specificity, sensitivity and throughput. This research presents a novel approach that overcomes these limitations using antibody-oligonucleotide conjugates for immuno-polymerase chain reaction (immuno-PCR) or proximity ligation, coupled with competitive PCR and MALDI-TOF mass spectrometry. Employing these combinations of technologies, we demonstrate multiplex detection and quantification of up to eight proteins, spanning wide dynamic ranges from femtomolar concentrations, using only microliter sample volumes.
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| 20. |
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| 21. |
- Giang, Kim Anh, et al.
(författare)
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Affibody-based hBCMA x CD16 dual engagers for NK cell-mediated killing of multiple myeloma cells
- 2023
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 77, s. 139-148
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Tidskriftsartikel (refereegranskat)abstract
- We describe the development and characterization of the (to date) smallest Natural Killer (NK) cell re-directing human B Cell Maturation Antigen (hBCMA) x CD16 dual engagers for potential treatment of multiple myeloma, based on combinations of small 58 amino acid, non-immunoglobulin, affibody affinity proteins. Affibody molecules to human CD16a were selected from a combinatorial library by phage display resulting in the identification of three unique binders with affinities (KD) for CD16a in the range of 100 nM–3 µM. The affibody exhibiting the highest affinity demonstrated insensitivity towards the CD16a allotype (158F/V) and did not interfere with IgG (Fc) binding to CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding arms, including bi-paratopic affibody combinations, were genetically fused to a high-affinity hBCMA-specific affibody. Such 15–23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines. Hence, we report a novel class of uniquely small NK cell engagers with specific binding properties and potent functional profiles.
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| 22. |
- Grimm, Sebastian, et al.
(författare)
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Monitored whole gene in vitro evolution of an anti-hRaf-1 affibody molecule towards increased binding affinity
- 2011
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 29:5, s. 534-542
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Tidskriftsartikel (refereegranskat)abstract
- The use of library technologies for the generation of affinity proteins often includes an affinity maturation step, based on the construction of secondary libraries from which second generation variants with improved affinities are selected. Here, we describe for the first time the affinity maturation of affibody molecules based on step-wise in vitro molecular evolution, involving cycles of error-prone PCR (epPCR) amplification for the introduction of diversity over the entire 58-residue three-helix bundle structure and ribosome display (RD) for the selection of improved variants. The model affibody molecule for the process was Z(RAF322), binding with a 1.9μm equilibrium dissociation constant (K(D)) to human Raf-1 (hRaf-1), a protein kinase of central importance in the MAPK/ERK proliferation pathway. The molecular evolution process was followed on both gene and protein levels via DNA sequencing and a biosensor-based binding analysis of pools of selected variants. After two cycles of diversification and selection, a significant increase in binding response of selected pools was seen. DNA sequencing showed that a dominant alanine to valine substitution had been effectively enriched, and was found in 83% of all selected clones, either alone or in combination with other enriched substitutions. The evolution procedure resulted in variants showing up to 26-fold increases in affinity to the hRaf-1 target. Noteworthy, for the two variants showing the highest affinities, substitutions were also found in affibody framework positions, corresponding to regions of the protein domain not addressed by traditional affibody molecule affinity maturation strategies. Interestingly, thermal melting point (T(m)) analyses showed that an increased affinity could be associated with both higher and lower T(m) values. All investigated variants showed excellent refolding properties and selective binding to hRaf-1, as analysed using a multiplexed bead-based binding assay, making them potentially valuable affinity reagents for cell biology studies.
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| 23. |
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| 24. |
- Gu, Gucci Jijuan, et al.
(författare)
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Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation
- 2013
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 30:2, s. 144-152
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Tidskriftsartikel (refereegranskat)abstract
- While antibodies currently play a dominant role as affinity reagents in biological research and for diagnostics, a broad range of recombinant proteins are emerging as promising alternative affinity reagents in detection assays and quantification. DNA-mediated affinity-based assays, such as immuno-PCR and proximity ligation assays (PLA), use oligonucleotides attached to affinity reagents as reporter molecules. Conjugation of oligonucleotides to affinity reagents generally employs chemistries that target primary amines or cysteines. Because of the random nature of these processes neither the number of oligonucleotides conjugated per molecule nor their sites of attachment can be accurately controlled for affinity reagents with several available amines and cysteines. Here, we present a straightforward and convenient approach to functionalize recombinant affinity reagents for PLA by expressing the reagents as fusion partners with SNAP protein tags. This allowed us to conjugate oligonucleotides in a site-specific fashion, yielding precisely one oligonucleotide per affinity reagent. We demonstrate this method using designed ankyrin repeat proteins (DARPins) recognizing the tumor antigen HER2 and we apply the conjugates in different assay formats. We also show that SNAP or CLIP tags expressed as fusion partners of transfected genes, allow oligonucleotide conjugations to be performed in fixed cells, with no need for specific affinity reagents. The approach is used to demonstrate induced interactions between the fusion proteins FKBP and FRB by allowing the in situ conjugated oligonucleotides to direct the production of templates for localized rolling circle amplification reactions.
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| 25. |
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| 26. |
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| 27. |
- Gustavsson, Elin, et al.
(författare)
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Surrogate antigens as targets for proteome-wide binder selection
- 2011
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 28:4, s. 302-311
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Tidskriftsartikel (refereegranskat)abstract
- In the last decade, many initiatives have been taken to develop antibodies for proteome-wide studies, as well as characterization and validation of clinically relevant disease biomarkers. Phage display offers many advantages compared to conventional antibody generation by immunization and hybridoma technology, since it is an unlimited resource of affinity reagents without batch-to-batch variation and is amendable for high throughput. One of the major bottlenecks to proteome-wide binder selection is the limited supply of suitable target antigens representative of the human proteome. Here, we provide proof of principle of using easily accessible, cancer-associated protein epitope signature tags (PrESTs), routinely generated within the Human Protein Atlas project, as surrogate antigens in phage selectionsfor the retrieval of target specific binders. These binders were subsequently tested in western blot, immunohistochemistry and protein microarray application to demonstrate their functionality.
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| 28. |
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| 29. |
- Heimersson, Sara, 1984, et al.
(författare)
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Methodological issues in life cycle assessment of mixed-culture polyhydroxyalkanoate production utilising waste as feedstock
- 2014
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 31:4, s. 383-393
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Tidskriftsartikel (refereegranskat)abstract
- Assessing the environmental performance of emerging technologies using life cycle assessment (LCA) can be challenging due to a lack of data in relation to technologies, application areas or other life cycle considerations, or a lack of LCA methodology that address the specific concerns. Nevertheless, LCA can be a valuable tool in the environmental optimisation in the technology development phase. One emerging technology is the mixed-culture production of polyhydroxyalkanoates (PHAs). PHA production by pure microbial cultures has been developed and assessed in several LCAs during the previous decade. Recent developments within mixed-culture PHA production call for environmental assessment to guide in technology development. Mixed-culture PHA production can use the organic content in wastewater as a feedstock; the production may then be integrated with wastewater treatment (WWT) processes. This means that mixed-culture PHA is produced as a by-product from services in the WWT.This article explores different methodological challenges for LCA of mixed-culture PHA production using organic material in wastewater as feedstock.LCAs of both pure- and mixed-culture PHA production were reviewed. Challenges, similarities and differences when assessing PHA production by mixed- or pure-cultures were identified and the resulting implications for methodological choices in LCA were evaluated and illustrated, using a case study with mixed- and pure-culture PHA model production systems, based on literature data.Environmental impacts of processes producing multiple products or services need to be allocated between the different products or services. Such situations occur both in feedstock production and when the studied system is providing multiple functions. The selection of allocation method is shown to determine the LCA results. The type of data used, for electricity in the energy system, is shown to be important for the results, which indicates, a strong regional dependency of results for systems with electricity use as an environmental hot spot. The importance of assessing water use, an environmental impact not assessed by any of the reviewed studies, is highlighted. © 2013 Elsevier B.V.
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| 30. |
- Hu, Francis Jingxin, 1986-, et al.
(författare)
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Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders
- 2018
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Ingår i: New Biotechnology. - : Elsevier. - 1871-6784 .- 1876-4347. ; 45, s. 80-88
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Tidskriftsartikel (refereegranskat)abstract
- Surface display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various display systems available, phage display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive bacterial host for display of a library of human antibody genes which, when combined with phage display, provides ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying low nanomolar affinity scFv fragments towards human epidermal growth factor receptor 2 (HER2). The ranking and performance of the scFv isolated by flow sorting in surface-immobilised form was retained when expressed as soluble scFv and analysed by biolayer interferometry, as well as after expression as full-length antibodies in mammalian cells. We also demonstrate the possibility of using Gram-positive bacterial display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. This combined approach has the potential for a more complete scan of the antibody repertoire and for affinity maturation of human antibody formats.
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| 31. |
- Hu, Francis Jingxin, et al.
(författare)
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Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody
- 2014
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 31:1, s. 35-43
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Tidskriftsartikel (refereegranskat)abstract
- One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues.
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| 32. |
- Hu, Francis Jingxin, 1986-, et al.
(författare)
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Phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders
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Ingår i: New Biotechnology. - 1871-6784 .- 1876-4347.
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Surface display couples genotype with a surface exposed phenotype and thereby allows for screening of gene-encoded protein libraries for desired characteristics. Of the various display systems, phage display is by far the most popular, mainly thanks to its ability to harbor large library sizes. Here, we describe the first use of a grampositive host for display of a library of human antibody genes. The method allows for swift generation of binders by combining phage and gram-positive display, for its ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying specific low nanomolar scFv towards human HER2. The ranking and performance of the scFv isolated by flow sorting in surface immobilized form was retained when expressed as soluble scFv and analyzed by biolayer interferometry as well as after expression as full-length antibodies in mammalian cells. We also show the possibility to use gram-positive display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. We believe this combined approach has the potential for a more complete scan of the antibody repertoire and for swift affinity maturation of human antibody formats.
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| 33. |
- Häggmark, Anna, et al.
(författare)
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Classification of protein profiles from antibody microarrays using heat and detergent treatment.
- 2011
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 29:5, s. 564-570
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Tidskriftsartikel (refereegranskat)abstract
- Antibody microarrays offer new opportunities for exploring the proteome and to identify biomarker candidates in human serum and plasma. Here, we have investigated the effect of heat and detergents on an antibody-based suspension bead array (SBA) assay using polyclonal antibodies and biotinylated plasma samples. With protein profiles from more than 2300 antibodies generated in 384-plex antibody SBAs, three major classes of heat and detergent susceptibility could be described. The results show that washing of the beads with SDS (rather than Tween) after target binding lowered intensity levels of basically all profiles and that about 50% of the profiles appeared to be lowered to a similar extent by heating of the sample. About 33% of the profiles appeared to be insensitive to heat treatment while another 17% showed a positive influence of heat to yield elevated profiles. The results suggest that the classification of antibodies is driven by the molecular properties of the antibody-antigen interaction and can generally not be predicted based on protein class or Western blot data. The experimental scheme presented here can be used to systematically categorize antibodies and thereby combine antibodies with similar properties into targeted arrays for analysis of plasma and serum.
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| 34. |
- Jakobsen, L., et al.
(författare)
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Functional proteomics of the human centrosome
- 2010
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Ingår i: New Biotechnology. - : ELSEVIER SCIENCE BV. - 1871-6784 .- 1876-4347. ; 27, s. S82-S82
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 35. |
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| 36. |
- Johnsson, Ola, et al.
(författare)
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A novel feeding strategy for industrial fed-batch processes based on frequency content analysis
- 2012
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 29:Supplement, s. 11-11
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Konferensbidrag (refereegranskat)abstract
- Overflow metabolism, i.e. the production of metabolic by-products at a high glycolytic flux, is a recurring problem in fed-batch processes with many types of microorganisms. In the current study, a novel feeding strategy aimed at avoiding process failures due to overflow by-product formation was designed and implemented in a pilot-scale reactor (0.5 m3). The basic principle behind the strategy was to analyze the effects on the dissolved oxygen concentration by periodic variations in the inlet feed rate. The frequency spectrum of the dissolved oxygen signal was used to estimate the proximity of the system to the region where overflow metabolism occurs by examining the content in the relevant frequency range. A control variable based on the measured frequency content was subsequently used to control the feed rate. The only measurement required for this strategy is the dissolved oxygen level in the broth, for which robust, fast and precise probes are widely available in industrial fermentors today. The strategy was successfully implemented in pilot-scale processes for industrial enzyme production using Bacillus licheniformis. It was shown possible to run the process close to the optimal feed rate, indicated by very low amounts of acetate (the overflow metabolite) in the broth. In comparison to a reference strategy the new control strategy resulted in over 10% higher biomass yields.
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| 37. |
- Jönsson, Malin, et al.
(författare)
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CaRA – A multi-purpose phage display library for selection of calcium-regulated affinity proteins
- 2022
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Ingår i: New Biotechnology. - : Elsevier B.V.. - 1871-6784 .- 1876-4347. ; 72, s. 159-167
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Tidskriftsartikel (refereegranskat)abstract
- Protein activity regulated by interactions with metal ions can be utilized for many different purposes, including biological therapies and bioprocessing, among others. Calcium ions are known to interact with the frequently occurring EF-hand motif, which can alter protein activity upon binding through an induced conformational change. The calcium-binding loop of the EF-hand motif has previously been introduced into a small protein domain derived from staphylococcal Protein A in a successful effort to render antibody binding dependent on calcium. Presented here, is a combinatorial library for calcium-regulated affinity, CaRA, based on this domain. CaRA is the first alternative scaffold library designed to achieve novel target specificities with metal-dependent binding. From this library, several calcium-dependent binders could be isolated through phage display campaigns towards a set of unrelated target proteins (IgE Cε3-Cε4, TNFα, IL23, scFv, tPA, PCSK9 and HER3) useful for distinct applications. Overall, these monomeric CaRA variants showed high stability and target affinities within the nanomolar range. They displayed considerably higher melting temperatures in the presence of 1 mM calcium compared to without calcium. Further, all discovered binders proved to be calcium-dependent, with the great majority showing complete lack of target binding in the absence of calcium. As demonstrated, the CaRA library is highly capable of providing protein-binding domains with calcium-dependent behavior, independent of the type of target protein. These binding domains could subsequently be of great use in gentle protein purification or as novel therapeutic modalities.
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| 38. |
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| 39. |
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| 40. |
- Klingström, Tomas, et al.
(författare)
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Workshop on laboratory protocol standards for the molecular methods database
- 2013
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 30:2, s. 109-113
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Tidskriftsartikel (refereegranskat)abstract
- Management of data to produce scientific knowledge is a key challenge for biological research in the 21st century. Emerging high-throughput technologies allow life science researchers to produce big data at speeds and in amounts that were unthinkable just a few years ago. This places high demands on all aspects of the workflow: from data capture (including the experimental constraints of the experiment), analysis and preservation, to peer-reviewed publication of results. Failure to recognise the issues at each level can lead to serious conflicts and mistakes; research may then be compromised as a result of the publication of non-coherent protocols, or the misinterpretation of published data. In this report, we present the results from a workshop that was organised to create an ontological data-modelling framework for Laboratory Protocol Standards for the Molecular Methods Database (MolMeth). The workshop provided a set of short- and long-term goals for the MolMeth database, the most important being the decision to use the established EXACT description of biomedical ontologies as a starting point.
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| 41. |
- Landegren, Ulf, et al.
(författare)
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A myopic perspective on the future of protein diagnostics
- 2018
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Ingår i: New Biotechnology. - : ELSEVIER SCIENCE BV. - 1871-6784 .- 1876-4347. ; 45, s. 14-18
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Forskningsöversikt (refereegranskat)abstract
- Plasma proteome analyses of the future promise invaluable insights into states of health, not only by measuring proteins whose role it is to ensure blood homeostasis, but increasingly also as a window into the health of practically any tissue in the body via so-called leakage protein biomarkers. Realizing more of this vast potential will require progress along many lines. Here we discuss the main ones, such as optimal selection of target proteins, affinity reagents, immunoassay formats, samples, and applications, with a view from ongoing work in our laboratory.
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| 42. |
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| 43. |
- Landegren, Ulf
(författare)
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Offshoots of the ESF functional genomics programme
- 2013
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 30:3, s. 296-298
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Tidskriftsartikel (refereegranskat)abstract
- After the conclusion of the second five-year period of the European Science Foundation (ESF) programme on functional genomics, it is time to take stock and evaluate its accomplishments. The programme networked leading scientists from a large number of European countries for strategy discussions about the promotion of functional genomics research, and to arrange scientific meetings and exchange programmes. In brief, I believe this programme has punched above its weight, and that it has successfully contributed to the overall organisation of molecular biosciences in Europe. With a modest annual budget the programme has created several interesting new opportunities, some of which may have yet to show their full impact. However, these mini-reviews are intended to provide a personal perspective on this functional genomics effort, and accordingly I focus on my personal experiences from the ESF programme.
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| 44. |
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| 45. |
- Leino, Mattias, 1989-, et al.
(författare)
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Purification of DNA oligonucleotides to improve hybridization chain reaction performance
- 2023
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Ingår i: New Biotechnology. - : Elsevier. - 1871-6784 .- 1876-4347. ; 76, s. 33-40
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Tidskriftsartikel (refereegranskat)abstract
- Hybridization chain-reaction (HCR) is technique to generate a linear polymerization of oligonucleotide hairpins, used in multiple molecular biology methods. The HCR reaction is dependent on that every hairpin is metastable in the absence of a triggering oligonucleotide and that every hairpin can continue the polymerization, which places a strong demand on oligonucleotide quality. In this paper we show how further purification can greatly increase polymerization potential. We found that a single extra PAGE-purification could greatly enhance hairpin polymerization both in solution and in situ. Purification using a ligation-based method further improved polymerization, yielding in situ immunoHCR stains at least 3.4-times stronger than non-purified control. This demonstrates the importance of not only good sequence design of the oligonucleotide hairpins, but also the demand for high quality oligonucleotides to accomplish a potent and specific HCR.
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| 46. |
- León-Vaz, Antonio, et al.
(författare)
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Exploring Nordic microalgae as a potential novel source of antioxidant and bioactive compounds
- 2023
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Ingår i: New Biotechnology. - : Elsevier. - 1871-6784 .- 1876-4347. ; 73, s. 1-8
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Tidskriftsartikel (refereegranskat)abstract
- Nordic microalgae are a group of photosynthetic organisms acclimated to growth at low temperature and in varying light conditions; the subarctic climate offers bright days with moderate temperatures during summer and cold and dark winter months. The robustness to these natural stress conditions makes the species interesting for large-scale cultivation in harsh environments and for the production of high-value compounds. The aim of this study was to explore the ability of nineteen species of Nordic microalgae to produce different bioactive compounds, such as carotenoids or polyphenols. The results showed that some of these strains are able to produce high amounts of carotenoids (over 12 mg·g-1 dry weight) and phenolic compounds (over 20 mg GAE·g-1 dry weight). Based on these profiles, six species were selected for cultivation under high light and cold stress (500 μmol·m-2·s-1 and 10 ˚C). The strains Chlorococcum sp. (MC1) and Scenedesmus sp. (B2–2) exhibited similar values of biomass productivity under standard or stress conditions, but produced higher concentrations of carotenoids (an increase of 40% and 25%, respectively), phenolic compounds (an increase of 40% and 30%, respectively), and showed higher antioxidant capacity (an increase of 15% and 20%, respectively) during stress. The results highlight the ability of these Nordic microalgae as outstanding producers of bioactive compounds, justifying their cultivation at large scale in Nordic environments.
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| 47. |
- Löfdahl, Per-Åke, 1959-, et al.
(författare)
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Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment complementation assay
- 2009
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Ingår i: New Biotechnology. - Amsterdam : Elsevier. - 1871-6784 .- 1876-4347. ; 26:5, s. 251-259
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Tidskriftsartikel (refereegranskat)abstract
- Protein fragment complementation assays (PCAs) based on different reporter proteins have been described as powerful tools for monitoring dynamic protein-protein interactions in living cells. The present study describes the construction of a PCA system based on genetic splitting of TEM-1 beta-lactamase for the selection of proteins specifically interacting in the periplasm of Escherichia coli bacterial cells, and its application for the selection of affibody molecules binding human tumour necrosis factor-alpha (TNF-alpha) from a combinatorial library. Vectors encoding individual members of a naive 10(9) affibody protein library fused to a C-terminal fragment of the beta-lactamase reporter were distributed via phage infection to a culture of cells harbouring a common construct encoding a fusion protein between a non-membrane anchored version of a human TNF-alpha target and the N-terminal segment of the reporter. An initial binding analysis of 29 library variants derived from surviving colonies using selection plates containing ampicillin and in some cases also the P-lactamase inhibitor tazobactam, indicated a stringent selection for target binding variants. Subsequent analyses showed that the binding affinities (K(D)) for three selected variants studied in more detail were in the range 14-27 nm. The selectivity in binding to TNF-alpha for these variants was further demonstrated in both a cross-target PCA-based challenge and the specific detection of a low nm concentration of TNF-alpha spiked into a complex cell lysate sample. Further, in a biosensor-based competition assay, the binding to TNF-alpha of three investigated affibody variants could be completely blocked by premixing the target with the therapeutic monoclonal antibody adalimumab (Humira (R)), indicating overlapping epitopes between the two classes of reagents. The data indicate that beta-lactamase PICA is a promising methodology for stringent selection of binders from complex naive libraries to yield high affinity reagents with selective target binding characteristics.
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| 48. |
- Ma, Qian, et al.
(författare)
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New fluorescently labeled auxins exhibit promising anti-auxin activity
- 2019
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 48, s. 44-52
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Tidskriftsartikel (refereegranskat)abstract
- The plant hormone auxin is a key player in the regulation of plant growth and development. Despite numerous studies devoted to understanding its role in a wide spectrum of physiological processes, full appreciation of its function is linked to a comprehensive determination of its spatio-temporal distribution, which plays a crucial role in its mode of action. Conjugation of fluorescent tracers to plant hormones enables sensitive and specific visualization of their subcellular and tissue-specific localization and transport in planta, which represents a powerful tool for plant physiology. However, to date, only a few fluorescently labeled auxins have been developed. We report the synthesis of four novel fluorescently labeled derivatives of indole-3-acetic acid (IAA) in the form of a conjugate with a nitrobenzoxadiazole (NBD) fluorophore together with validation of their biological activity. These compounds, unlike other previously reported auxins fluorescently labeled at N1 position (nitrogen of the indole ring), do not possess auxin activity but rather show dose-dependent inhibition of auxininduced effects, such as primary root growth inhibition, root hair growth and the auxin reporter DR5::GUS expression. Moreover, the study demonstrates the importance of the character of the linker and optimal choice of the labeling site in the preparation of fluorescently labeled auxins as important variables influencing their biological activity and fluorescent properties.
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| 49. |
- Mandenius, Carl-Fredrik, et al.
(författare)
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Mechatronic design methodology for biotechnology products
- 2009
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Ingår i: New Biotechnology. - Amsterdam : Elsevier. - 1871-6784 .- 1876-4347. ; 25:Suppl. 1, s. S190-S190
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Biotechnology products can be divided into (1) biologics, which comprise those metabolites, biopolymers, and cell structures that are produced through biological processes, and (2) biotechnical machines, which are apparatuses and devices that transform, change, or analyze ‘biological specimens’ for specific purposes, often by using the biological systems per se. The first category has been thoroughly treated in bioengineering theory and practice while the second has been very scarcely investigated.In this presentation is described how the general design science theory can be applied when designing a technical system where biological species or components have the key role in the engineering design solutions. We have named these systems bio-mechatronic systems, since they are combined design achievements between traditional electronic and mechanical sub-systems and the biological systems, and where biological molecules and/or active microbial or cellular components influence the design solutions in a complex way.The purpose is to demonstrate that biotechnology and bioengineering related design can utilize and benefit from other commonly used design tools in, for example, mechanical and electric engineering. These tools should result in shorter development times and a reduction of the need for prototype testing and verification.Four examples will be presented, all well-known biotechnology products in the pharmaceutical and clinical areas: (1) a protein purification system, (2) a bioreactor system, (3) a biosensor instrument, and (4) an embryonic stem cell manufacturing systems.
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| 50. |
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